Triglycerides in Oils Analysis by the 5975-SMB GC-MS with Cold EI

Aviv Amirav, Tel Aviv University and Aviv Analytical, Tel Aviv Israel.
Hans-Gerd Janssen, Unilever R&D Vlaardingen and University of Amsterdam, Amsterdam The Netherland

Introduction 

Triglycerides analysis is challenging since these large compounds do not elute from standard GC columns under the conditions used in GC-MS and furthermore their standard EI mass spectra do not exhibit any molecular ion, as shown in the NIST library. In addition, their analysis by LC-MS is also challenging in view of their poor proton affinity. Thus, and in view of the importance of triglycerides analysis we decided to analyze three oil samples that were previously analyzed by GC-FID in order to evaluate these oils analysis by GC-MS with Cold EI. The challenge was to properly elute these large compounds and demonstrate the availability of abundant molecular ions plus useful and informative high mass fragments. Triglycerides analysis is very important for the food industry and such analysis can also be potentially important for human medical diagnostics in the form of cholesterol and triglycerides analysis in blood. Currently triglycerides are hydrolyzed and methylated to form FAMEs (fatty acid methyl esters) that are analyzed by GC-MS. However, in such analysis vital information is lost on the actual structure and concentration of the various parent triglycerides and GC-MS with Cold EI is challenged to exhibit such information.     

Pesticide Analysis by Pulsed Flow Modulation GCxGC-MS with Cold EI – An Alternative to GC-MS-MS

Uri Keshet (1), Tal Alon (1), Paulina Goldshlag (2) and Aviv Amirav (1)

1. School of Chemistry, Tel Aviv University, Tel Aviv, Israel. 

2. Israel Plant Protection and Inspection Services, Beit Dagan, Israel.

3. Aviv Analytical LTD, Tel Aviv, Israel.   

Introduction 

Pesticide analysis in agricultural products is an application area of growing importance which is usually addressed by GC-MS and LC-MS. However, pesticide analysis is confronted by the dominance of matrix interference which severely restricts the achievable limits of detection. As a result, several methods were developed and used for the reduction of matrix interference and the most widely used is MS-MS. While in LC-MS, MS-MS is practically a must in view of absence of libraries and consistent fragments in the mass spectra, in GC-MS the use of full scan possesses a few advantages of unambiguous library identification and its applicability to unlimited number of GC-MS amenable pesticides, provides however that the problem of matrix interference can be properly addressed. In addition to MS-MS at least three other techniques can be used to reduce matrix interference while enabling the use of full scan namely: a) high resolution mass spectrometry; b) GC-MS with Cold EI in which the enhanced molecular ions suppress matrix interference as described in another article in this blog. c) GCxGC-MS with its improved GC separation that can be used for the reduction of matrix interference as described in a few papers with thermal modulation and as discussed in this article.  

We developed Pulsed Flow Modulation (PFM) for obtaining GCxGC-MS with supersonic molecular beams (SMB) (also named Cold EI) based on a low cost single quadrupole mass analyzer and without any added cooling gas. For further information on the concept of Pulsed Flow Modulation GCxGC please read M. Poliak, M. Kochman and A. Amirav, "Pulsed Flow Modulation Comprehensive Two Dimensional Gas Chromatography" J. Chromatogr. A. 1186, 189-195 (2008) (also Israel patent number 176724 and USA patent number 7518103). 

In PFM-GCxGC-MS with Cold EI, sample compounds which elute from the first GC column are temporarily stored in a fused silica transfer line and are pulsed periodically injected by ~25 ml/min He gas pulse into the second column. After the pulse, ~20 ml/min He develops the chromatography in the second column for a few seconds (typically 4s). PFM is simple to construct, does not require any added gas with Cold EI and the injection time can be tuned, hence PFM-GCxGC-MS with Cold EI is enabled with quadrupole MS. The increased PFM second GC column flow rate is seamlessly handled by the Cold EI nozzle vacuum chamber.

We combined PFM-GCxGC with the Aviv Analytical 5975-SMB GC-MS with Cold EI and analyzed with it twelve pesticides in two agricultural matrices. PFM-GCxGC-MS with Cold EI combines the improved separation of GCxGC with the features of Cold EI of enhanced molecular ions and isotope information for the provision of ultimate sample identification information. Unique to PFM-GCxGC-MS with Cold EI is that in contrast to thermal modulation with standard GC-MS the second dimension elution time does not suffer from any ion source peak tailing and/or second column saturation effects and thus it is ideally suitable for pesticide analysis in complex matrices.   

Keep reading to find out how the Aviv Analytical 5975-SMB GC-MS with Cold EI enables effective pesticide analysis in agricultural matrices while using improved PFM-GCxGC separation for obtaining a major reduction of matrix interference by an average measured factor of 32. As a result, the road is open for PFM-GCxGC-MS with Cold EI to serve as an alternative to GC-MS-MS while enabling the use of full scan which is applicable to unlimited number of GC-MS amenable pesticides with improved library identification and the novel use of isotopomer ratios for pesticides identity confirmation

Drug Impurities Analysis by the Aviv Analytical 5975-SMB GC-MS with Cold EI

Aviv Amirav, School of Chemistry, Tel Aviv University and Director – Aviv Analytical 

Drugs are typically analyzed by LC-MS. When active pharmaceutical ingredients (API) are analyzed for the presence of impurities, the general requirements are that every impurity found should be at concentration below 0.1% of the API or it must be either cleaned or identified and toxicologically characterized which is a time consuming and expensive procedure. However, the ionization yields of Electrospray and/or APCI in LC-MS are highly non-uniform and compound dependent, and as a result when an impurity is found in a given LC-MS mass chromatogram its actual concentration is not known. Consequently, regardless of its actual concentration that can be well below 0.1% every impurity that is found must be isolated, fully identified, synthesized (or obtained) and serve for the provision of a calibration curve in order to measure the actual concentration of that small impurity peak. In addition, several types of impurities such as non-polar compounds are not properly ionized and are not detected by LC-MS. Thus, clearly drug impurity analysis represents an unmet analytical challenge.  

Alternatively, GC-MS with its standard electron ionization (EI) can be used and has, for volatile compounds, approximately uniform, semi-quantitative ionization yield. However, GC-MS with standard EI is compatible only with a limited range of thermally stable drugs and it provides useful molecular ions only for a portion of the analyzed compounds. Furthermore, ion source degradation and peak tailing severely erode the standard EI response uniformity for semi-volatile compounds. 

We used the Aviv Analytical 5975-SMB GC-MS with Cold EI for the analysis of drug impurities. Cold EI is the electron ionization of cold molecules in supersonic molecular beams (SMB). The technology of GC-MS with Cold EI is reviewed by A. Amirav, A. Gordin, M. Poliak, and A. B. Fialkov, J. Mass Spectrom., 43, 141-163. (2008). The later is available upon request. 

GC-MS with Cold EI as implemented with the Aviv Analytical 5975-SMB uniquely extends the range of drugs that are amenable for GC-MS analysis, including thermally labile drugs that are not compatible with standard GC-MS analysis. Furthermore, it is characterized by having uniform response to all compounds due to its contact-free fly-through ion source that eliminates any ion source peak tailing or losses. In addition, it provides trustworthy enhanced molecular ions that can be converted with the Tal-Aviv Molecule Identifier software into elemental formulae. Thus, the high sensitivity and uniform response of Cold EI uniquely enables the detection, identification and quantitation of low level impurities in drugs without lengthy calibration procedures. 

In order to evaluated the above attributes of the 5975-SMB we analyzed impurities in two Active Pharmaceutical Ingredient (API) samples (powder form) of Haloperidol and Ramelteon (Structures are given below).   

      

 

Keep reading to find our how the Aviv Analytical 5975-SMB GC-MS with Cold EI uniquely enables quantitative drug impurities analysis and helps in their identification. 

A Universal Method for Pesticide Analysis with the 5975-SMB GC-MS with Cold EI

 Aviv Amirav, Professor of Chemistry at Tel Aviv University and Director – Aviv Analytical

Introduction 

An estimated 2000 chemicals are being used as pesticides worldwide, including banned pesticides and other hazardous chemicals. In view of international food trade this large number implies that pesticide analysis should not be treated as target compounds analysis. Thus, current mass spectrometry instrument development is challenged to provide a one system and method that will be capable of analyzing as many pesticides as possible in full scan mode, in the needed instrumental sensitivity and selectivity in complex agricultural matrices, and in a short amount of time for effective (and lower cost) pesticides screening.   

In order to advance towards meeting the above challenges, a GC-MS with Cold EI (EI of Cold Molecules in Supersonic Molecular Beams) was evaluated in the past with pesticide analysis in agricultural products as described in M. Kochman, A. Gordin, P. Goldshlag, S. J. Lehotay and A. Amirav "Fast, High Sensitivity, Multi-Pesticide Analysis of Complex Mixtures with the Supersonic GC-MS" J. Chromatogr. A. 974, 185-212 (2002). Recently, we further evaluated pesticide analysis with the Aviv Analytical 5975-SMB GC-MS with Cold EI (GC-MS with supersonic molecular beam (SMB) interface and its fly through ion source) and developed a method to meet the above challenges. 

Keep reading to find out how the Aviv Analytical 5975-SMB GC-MS with Cold EI enables a truly universal method of pesticide analysis in agricultural matrices with improved range of compounds amenable for analysis, reduced matrix interference, lower LOD particularly for difficult to analyze pesticides, improved pesticide identification and shorter analysis time.   

Selectivity Enhancement for the Reduction of Matrix Interference in GC-MS – Cold EI as an Alternative to MS-MS

Aviv Amirav, Professor of Chemistry at Tel Aviv University and Director – Aviv Analytical, 

Introduction 

Matrix interference is often the bottleneck in achieving low limits of detection (LOD) when the sample compounds are in complex matrices such as in the analysis of pesticides in various agricultural products. As a result, advanced ways of selectivity enhancement for the reduction of matrix interference were developed, such as MS-MS and high resolution MS. In MS-MS, the selected parent ions undergo collision induced dissociation and one or two daughter ions are transmitted and detected in the third quadrupole MS. We estimate that MS-MS involves with a signal loss of about a factor of 10 while the matrix signal is reduced by an average factor of 200 thereby the selectivity against matrix interference is improved by a factor of about 20. In high resolution MS the selectivity gain is about the resolution/1000 while for compounds with negative mass defects such as dioxins and other multi halogen compounds it can be much higher. Nevertheless, high resolution MS also suffers from reduced signal compared to single ion monitoring with a simple single quadrupole MS instrument. The topics of selectivity gain in MS-MS and high resolution MS will be separately discussed in future blog posts. 

In this post we wish to illuminate another option to reduce matrix interference which is the use of Cold EI (EI of cold molecules in Supersonic Molecular Beams (SMB)) with its enhanced molecular ions, and compare it with MS-MS. 

It is known that matrix interference is reduced with mass. We found that the matrix interference reduction factor is about twenty per one hundred amu higher mass. (M. Kochman, A. Gordin, P. Goldshlag, S. J. Lehotay and A. Amirav "Fast, High Sensitivity, Multi-Pesticide Analysis of Complex Mixtures with the Supersonic GC-MS" J. Chromatogr. A. 974, 185-212 (2002)). Thus, the molecular ion is by far the most selective ion in the EI mass spectrum against matrix interference. As a result, we claim that SIM on the enhanced molecular ion in Cold EI is about as selective against matrix interference as MS-MS on a fragment ion as a parent ion. Since most pesticides are analyzed by MS-MS with standard EI on their major fragment ions as parent MS-MS ions (since their molecular ions are weak or absent), we claim that the Aviv Analytical 5975-SMB GC-MS with Cold EI is as selective as GC-MS-MS with standard EI. 

Keep reading to find out how the Aviv Analytical 5975-SMB GC-MS with Cold EI enables significant reduction in matrix interference while unlike MS-MS it further enables simultaneous full scan and SIM analysis for achieving universal pesticide screening.  

Glycidyl Esters Analysis by the Aviv Analytical 5975-SMB GC-MS with Cold EI

Aviv Amirav, Professor of Chemistry at Tel Aviv University and Director - Aviv Analytical

Introduction

Glycidyl fatty acid esters (Glycidyl Esters or GE in short) have the general structure as shown for Glysydyl stearate below.

Glysydyl Stearate

Glycidyl esters are of concern as they may occur in refined edible fats and oils and corresponding finished food products, including infant formula. The toxicological relevance of glycidyl esters is just as unclear as the question of their hydrolysis within the human digestive tract. Glycidol itself – the epoxide of glycerol - has carcinogenic and mutagenic features. The German Federal Institute for Risk Assessment (BFR) presently assumes that glycidol is fully released for glycidol esters in the digestive tract. In addition, Glydidyl esters are also linked with the appearance of the highly toxic 3-Monochloro-1,2-propanediol (3-MCPD) in various types of food, Thus, it is important to identify glycidyl esters in food. However, the analysis of Glycidyl esters can be problematic as they are known to be thermally labile and also having a weak or completely absent molecular ions in their EI mass spectra. At the recent 2012 Capillary Chromatography meeting in Riva del Garda, Sjaak de Koning, Herrald Steenbergen, and Hans-Gerd Janssen presented their work on the analysis of glycidyl esters while using the Leco Pegasus HT GC-TOF-MS. 

In their work, no molecular ion was observed as shown above. We believe that without having molecular ions one cannot and should not trust that these thermally labile compounds actually eluted as these mass spectra can correspond to injector or transfer line degradation or on-the-shelf degradation products. In addition, with abundant m/z=116 ion and weak m/z=185 ion in the EI mass spectra the selectivity of glydidyl esters analysis with such low mass fragment ions in complex food matrices is expected to be limited.

Thus, motivated by this challenge and after discussing it with Dr. Hans-Gerd Janssen from Unilever we decided to analyze glycidyl esters using the 5975-SMB GC-MS with Cold-EI, and our results are reported in this post.

Beeswax in Fruit and Vegetables Analysis and the Emerging Challenge of Material Identification

Aviv Amirav, Professor of Chemistry at Tel Aviv University and Director – Aviv Analytical 

Igal Bar Ilan Ph.D.  Migal Laboratories, Kiryat Shmona Israel    

Introduction 

Beeswax serves to treat growing numbers of fruits and vegetables in order to increase its shelf time, reduce product dehydration, suppress mold growth and improve the appearance (shiny) of the treated fruits or vegetables. However, while wax is generally considered as safe, some say that such treatment serves as an evidence for extended (long) time between harvest and consumption hence reduction in certain nutrients. A few types of waxes are used but beeswax is considered as an "organic" wax hence of high quality. Recently beeswax analysis is required in certain fruits and vegetables (by the EU) and standard GC-MS or LC-MS failed to perform such challenging analysis. Beeswax is used in combination with minor addition of olive oil and the challenge is to develop a method to analyze such wax in the 1-100 ppm range in certain fruits and vegetables. Note that wax is a mixture of several heavy compounds, most notably hydrocarbons and heavy esters plus some triglycerides, thus 1 ppm means about 40 ppb each of its major heavy ingredients. In addition, unlike in pesticide analysis in this case the emerging challenging need is to identify a material as opposed to compound. 

After method development we successfully analyzed the wax in 16 fruit and vegetable samples including avocado, cucumber, tomato and apple using the Aviv Analytical 5975-SMB GC-MS with Cold-EI.

Keep reading for the analysis details, results and conclusions.