News from LabRulezGCMS Library - Week 12, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezGCMS Library in the week of 17th March 2025? Check out new documents from the field of the gas phase, especially GC and GC/MS techniques!

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This week we bring you application notes by Agilent Technologies, Shimadzu and Thermo Fisher Scientific!

1. Agilent Technologies Hydrogen Carrier Gas for GC/MS/MS Analysis of Steroids in Urine in Under 10 Minutes

• Application note
• Full PDF for download

Anabolic-androgenic steroids (AAS) are a class of steroidal hormones related to testosterone. Testosterone is naturally produced in the human body and is primarily excreted as a conjugate with glucuronide or sulfate. AAS exhibit both androgenic effects, associated with masculinization and virilization, and anabolic effects, linked to protein synthesis and muscle growth. Despite the ongoing debate about their efficacy and potential risks, the use of AAS is prevalent in power sports for muscle mass enhancement and in endurance sports for improved recovery. Furthermore, various indirect steroid doping strategies can similarly elevate testosterone levels, complicating the detection and regulation of doping practices.

Endogenous AAS are all naturally present in the bodily fluids of both males and females. However, some endogenous AAS (for example, testosterone and DHEA) are also available as pharmaceutical drugs or marketed as "food supplements" in some countries. These drugs and supplements remain prohibited by WADA, but detecting their use is much more difficult due to the natural presence of endogenous AAS. Therefore, the detection of possible misuse is determined by evaluating AAS concentrations and ratios over a long period in the individual athlete's steroid passport. If significant fluctuations from the long-term mean appear, the sample can be forwarded to isotope ratio mass spectrometry (IRMS), which can unequivocally show the synthetic origin of the steroid. An alternative for IRMS analysis is the detection of steroid esters in the blood.

Triple quadrupole GC/MS/MS3 and high-resolution GC/Q-TOF4 are essential techniques, especially for the quantification of AAS and the separation of stereoisomers that LC/MS techniques often fail to resolve. Traditionally, helium (He) has been the carrier gas of choice for GC/MS analyses due to its optimal performance characteristics. However, recurring global helium shortages have necessitated the search for viable alternatives to ensure the continuity and reliability of analytical measurements. 

Hydrogen could offer several advantages over helium as a carrier gas in GC/MS, including the fact that it is more readily available and significantly less expensive than helium. Hydrogen can also be produced sustainably through the electrolysis of water using green electricity, making it a more environmentally friendly option. Additionally, hydrogen keeps the electron ionization (EI) source cleaner during operation, reducing maintenance needs and increasing instrument uptime and lab productivity. 

This application note presents a developed and validated GC/MS/MS method for the effective and reliable measurement of 14 endogenous AAS in urine, using electron ionization with hydrogen as the carrier gas. Method performance was maintained, meeting the same LOIs as the method using helium as the carrier gas. The feasibility of the transition to hydrogen as a carrier gas in GC/MS/MS applications provides antidoping laboratories with a robust alternative that ensures the continuity of high-quality analytical results in the face of helium supply challenges.

Experimental

GC/TQ analysis 

An Agilent 7000C triple quadrupole GC/MS (GC/TQ) system was used for the analysis. The instrument operating parameters are listed in Table 2. Additional considerations and best practices for using H2 as a carrier gas can be found in the Helium to Hydrogen Carrier Gas Conversion User Guide. 5 Given that this was a targeted analysis and none of the targets demonstrated in-source reactivity with hydrogen carrier gas, a standard Agilent Inert Plus EI source was used. For analysis of unknowns or when analyzing compounds that are prone to interacting with hydrogen, an Agilent HydroInert source can be considered.

The previous positive chemical ionization (PCI) GC/MS/MS method using helium as the carrier gas used a 5:1 split.6 However, it is common to observe a 2 to 5x decrease in sensitivity when converting the analysis to using hydrogen carrier gas. To compensate for decreased sensitivity, the injection split ratio or injection volume can be adjusted. In this newly developed method using hydrogen gas, splitless injection was employed to maintain sufficient sensitivity and comparability with the previous method. 

Data acquisition and processing were performed using Agilent MassHunter acquisition software for GC/MS systems (version 10.2) and Agilent MassHunter Quantitative Data Analysis software (version 10.2). 

Calibration curves for the 14 steroids included six points each and were prepared by spiking steroids into stripped urine. The calibration levels are provided in Table 3. Each calibration was run in triplicate, resulting in 18 calibration points at six levels. Deuterated internal standards (ISTDs) were used for internal standard calibration. Quadratic fit was used for all compound calibrations. The calibration curves had a 1/x weighting factor applied.

Results and discussion 

Quantitation of 14 steroids in urine Table 3 shows calibration ranges and correlation coefficients for 14 steroids that require quantitation according to WADA. The endogenous anabolic steroids quantitated in this work are some of the most analytically challenging compounds. On one side, this is due to their low LOIs, but on the other side, challenges can arise from the need to accurately quantify them at high concentrations. Using this method, the correlation coefficients (R2 ) were greater than 0.997 for all 14 targets. 

Figure 3 shows MRM chromatograms at the lowest calibration point for epitestosterone, testosterone, 6α-OH-androstenedione, androsterone, and etiocholanolone. At their lowest calibration points, all compounds have a signal-to-noise ratio greater than three. Additionally, androsterone and etiocholanolone are fully resolved at their lowest calibration levels. 

To illustrate observed peak fronting, chromatograms for androsterone and etiocholanolone at the highest calibration level are also shown (Figure 3, bottom). This peak fronting is a result of GC column overloading at 9,600 ng/mL. Despite the peak fronting, excellent calibrations with R2 values of 0.999 were achieved for both compounds.

Conclusion 

A GC/MS/MS method with electron impact ionization (EI) using hydrogen carrier gas was developed for the analysis of 14 anabolic steroids in urine. Excellent calibration performance was shown across an extended calibration range covering the minimum required performance levels (MRPLs). This analysis could be performed in under 10 minutes, which is 40% faster than the original positive chemical ionization (PCI) GC/MS/MS method. The WADA validation demonstrated that for all analyzed samples, z-scores were less than 1, indicating acceptable method accuracy.

2. Shimadzu: Analysis of 2-Methylisoborneol and Geosmin in Drinking Water by GC-MS and SPME Arrow

• Application note
• Full PDF for download

User Benefits

• SPME Arrow offers highly durable and sensitive trace analysis.
• Using SPME Arrow not only provides cost savings in the long run but also eliminates the need for the salting-out technique,
  contributing to overall cost efficiency.

2-methylisoborneol (2-MIB) and geosmin are responsible for earthy and musty odors which can affect the taste of drinking water. Hence, the analysis of these compoundsin watersourcesis important due to their significant impact on water quality and taste. As humans can perceive them at trace levels (ng/L)[1], it is crucial to detect them at this concentration. The Solid Phase Microextraction (SPME) Fiber technique paired with GC-MS has been used to detect these two organic compounds previously; however, salting-out is often required to improve sensitivity. Moreover, if the salt purity is insufficient, it may lead to contaminants co-eluting with the target compounds. This application news introduces a study utilizing GC-MS with SPME Arrow to effectively detect and quantitate the concentration of geosmin and 2-MIB in drinking water samples. Notably, salting-out is not required, and a limit of quantitation of 1 ng/L still can be achieved, with area %RSD (n=5) for 2-MIB and geosmin being less than 5 %. The spike recovery study at concentrations of 1 ng/L yielded recoveries within 70 % to 130 %.

Experimental 

Analysis condition 

The instruments utilized in this experiment were GCMS-QP2020 NX and AOC-6000 Plus (both from Shimadzu Corporation, Japan), as shown in Figure 1. SPME Arrow in AOC-6000 Plus, designed to enhance the extraction and analysis of volatile and semi-volatile compounds from various matrices, was selected as the sampling technique. This sampling method utilizes a larger adsorbent amount compared to the traditional SPME Fiber, allowing for increased sample capacity and improved sensitivity. SPME Arrow also possesses a thick and sturdy construction, which contributes to its durability and, consequently, cost saving in the long run

Conclusion 

With SPME Arrow technique paired with GC-MS, a method was successfully developed for the analysis of 2-MIB and geosmin in drinking water in trace level. Good sensitivity (LOQ at 1 ng/L), area repeatability and linearity (R² > 0.999) were achieved without using the salting-out technique. Good recovery at 1 ng/L was obtained (within 70 % to 130 %) for both compounds in drinking water samples.

3. Thermo Fisher Scientific: Workflow for differential analysis of whisky using an Orbitrap Exploris GC 240 mass spectrometer

• Application note
• Full PDF for download

Whisky is a premium spirit beverage that is distilled by following long-established methods that involve a complex aging process. It is produced by the mixing of various grains with water to form a mash that is fermented with yeast, distilled to generate an alcoholic distillate, and finally matured in wooden barrels or casks. This is a complex and traditional process that results in a beverage that has both a high value and high degree of variability. The production technology plays a significant role in the chemical composition and hence the flavor characteristics of the final whisky product. 

Gas chromatography-mass spectrometry (GC-MS) has been widely used to characterize whisky as it provides analytical advantages of chromatographic resolution, reproducibility, peak capacity, and, most importantly, extensive spectral libraries to aid in identification of volatile and semi-volatile chemical constituents. In this study, we take advantage of the performance of the Orbitrap Exploris GC 240 mass spectrometer for the profiling of whiskies of different origins, ages, and types. An additional aim is to evaluate the application of a complete untargeted chemometric workflow using Compound Discoverer software to detect and identify chemical components in whisky. It will also show the process of identifying chemical differences in whiskies of different origins. Samples were analyzed using a full scan non-targeted acquisition and high mass resolving power (120,000 resolution FWHM at m/z 200) to obtain accurate mass measurements. This is important to enable elucidation of the elemental composition and discrimination of co-eluting and isobaric compounds. 

These features in combination with unique software algorithms for automated deconvolution and sample comparison create a powerful solution for comprehensive characterization, quality control, and product brand protection.

In this study, the performance of the Orbitrap Exploris GC 240 high resolution accurate mass (HRAM) spectrometer together with the headspace solid phase micro extraction (SPME Arrow) for chemical profiling of whisky is demonstrated.1 Differences in chemical profiles are easily visualized using the statistical tools incorporated into Compound Discoverer software with streamlined identification using the Thermo Scientific™ Flavour and Fragrance HRAM library. This provides analysts with a means of accurate identification of chemical profiles in whisky to maintain product quality control and differentiate between whiskies suspected of fraudulent activities (additives and mislabeling).

Experimental

Instrument and method setup Headspace extraction and injection of whisky samples was performed using the Thermo Scientific™ TriPlus™ RSH SMART autosampler equipped with the Thermo Scientific™ SMART SPME Arrow 1.1 mm DVB/C-WR/PDMS fiber (P/N 36SA11T3-SM). Incubation and extraction were performed online followed by sample injection/desorption. After sample injection, the SPME Arrow fiber was re-conditioned at high temperature under a nitrogen flow using an SPME conditioning station to avoid sample carryover between injections. Further details surrounding the SPME Arrow operating parameters can be found in Table 1.

A Thermo Scientific™ TRACE™ 1610 GC equipped with a Thermo Scientific™ TraceGOLD™ TG-624SilMS (30 m × 0.25 mm I.D. × 1.4 µm film) capillary column (P/N 26085-3320) was used to perform the chromatographic separation. Oven program conditions can be found in Table 1. Data acquisition was carried out in full scan analysis mode using both EI and PCI with the Orbitrap Exploris GC 240 mass spectrometer. Additional MS method parameters are summarized in Tables 2 and 3. External mass calibration was performed prior to analysis, while characteristic ions representing column bleed were used as lock masses when scanning in EI to perform internal mass calibration. Sample acquisition and qualitative processing were performed using the Thermo Scientific™ Chromeleon™ version 7.3.2 Chromatography Data System (CDS) software. Unknown analysis and identification were performed using the Compound Discoverer version 3.3 software.

Results and discussion

Discovering differences between samples 

The first objective was to identify if there was any significant difference between the whisky samples. This was achieved through a PCA plot of the replicate injections of each sample. Figure 3 shows the PCA plot that demonstrates that there are clear differences in the identified chemical profiles between the samples and good agreement of the replicate injections, for example between samples 2 and 3. 

The following steps then help identify which peaks contribute to the differences so that a compound identification can be proposed. As an example, Figure 4 shows a volcano plot for sample 3 versus 2. The volcano plot is a type of scatter plot for replicate data where the x axis represents the log2 of the fold change between two sample groups (generated ratio), and the y axis represents the negative log10 of the p-value (test of significance) of the fold change. 

In other words, when a point (compound) is more on the left (positive values on x axis), the peak area of that compound is much higher in sample 2 than in sample 3, while points that are higher on the graph are statistically more significant. For example, higher levels of 2/3 methyl-1- butanol are typically associated with Scotch malt whiskey, as these are reduced in the grain whiskey due to the distillation processes within column stills. This is further supported by the higher Furfural content also found in sample 2, which is present in higher amounts in malted grains. High ester content is also associated with age and cask maturation (i.e., bourbon casks), indicating sample 2 has undergone a much longer and different maturation process compared to sample 3.)

Conclusion 

The complex and versatile chemical profile existing among the various whisky types causes challenges in sample profiling and finding marker compounds. A combination of the HS-SPME Arrow, Orbitrap Exploris GC 240 mass spectrometer, and Compound Discoverer software provides an efficient workflow for the analysis of compounds giving analytical advantages including:

• Time savings with minimal sample preparation and online extraction using the TriPlus RSH SMART robotic autosampler 
• Full scan acquisition at high mass resolution (i.e., 120,000 at m/z 200) providing targeted quantitative analysis together with non-target analysis for chemical profile determination 
• Mass spectral deconvolution combined with statistical tools for sample differentiation, all combined within the Compound Discover software 
• Dedicated Flavor and Fragrance HRAM library for accurate identification at sub ppm mass accuracy 

The workflow on food profiling in this application note can be applied to other areas that involve sample group comparison, screening, and compound identification.2-4