News from LabRulezLCMS Library - Week 20, 2026

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 11th May 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!

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

1. Agilent Technologies: Quantitation of Over 300 Pesticide Residues in Three Processed Cocoa Matrices

• Application note
• Full PDF for download

Despite low bean production locally, Malaysia ranks among the world's top cocoa grinders, transforming raw beans into powder, liquor, and butter (Malaysian Cocoa Board, 2023). These value-added products are major contributors to the country's exports. Traces of contamination, including pesticide residues, have been spotted in processed cocoa beans, necessitating thorough inspection. 

Processed cocoa beans pose complicated challenges for sample extraction and matrix removal. Cocoa liquor is in the form of pure, ground paste made using roasted cocoa beans, which when pressed, separate into fat (cocoa butter) and solids (cocoa powder). These matrices, the most common forms of processed cocoa beans, are used to conduct a comprehensive screening of pesticide residues. 

Extraction and cleanup methods were adopted from a cinnamon workflow¹, as cinnamon and cocoa beans exhibit similarities in terms of their complex, dry matrices. Due to their dark brown color, pigment removal is essential for the sample preparation of processed cocoa beans. The Agilent Bond Elut QuEChERS AOAC extraction kit was implemented for sample extraction and the Agilent Captiva EMR-GPD was used for highly selective and efficient cleanup. The cartridge was packed with the following sorbents: Carbon S for pigment removal, primary secondary amine (PSA) for fatty acid removal, and C18 for additional hydrophobic matrix removal.

Experimental

• LC: Agilent 1290 Infinity III LC
• Column: Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 × 150 mm, 1.8 µm
• MS: Agilent 6475 Triple Quadrupole LC/MS
• MS Ion Source: Agilent Jet Stream Technology Ion Source (AJS)

Conclusion 

Sensitive and robust analytical methods for screening over 300 pesticide residues in three processed cocoa matrices (powder, liquor, and butter) were developed using the Agilent 6475 triple quadrupole LC/MS. A fast and reliable extraction and passthrough cleanup method using the Agilent Bond Elut QuEChERS AOAC extraction kit and the Agilent Captiva EMR–GPD cartridge was evaluated for different cocoa matrices. A combination of effective extraction, selective matrix removal, and sensitive analytical measurement provides a comprehensive end-to-end solution for the downstream cocoa industry for screening pesticide residues in processed cocoa matrices.

2. Metrohm: IC-MS/MS analysis of trifluoroacetic acid according to DIN 38407-53

TFA: a pervasive PFAS breakdown product of increasing concern

• Application note
• Full PDF for download

Trifluoroacetic acid (TFA) is an ultra-short-chain PFAS (per- and polyfluoroalkyl substance) belonging to the most toxic group of perfluorooctanoic acids (PFOA) (Figure 1) [9]. Overall, TFA concentrations are significantly higher than those of other PFASs, as TFA is a breakdown product of those substances [3]. For example, TFA accounted for more than 90% of the measured total PFASs in various analyzed German drinking waters [9]. It has emerged as a ubiquitous and persistent substance with high mobility due to its lack of sorption, and raises several environmental and regulatory concerns [3–6,10]. TFA's ongoing emissions are causing irreversible concentration increases, for example, in rainwater, soils, and drinking water, but also in human serum, plants, and plant-based foods [3]. Measured median concentrations of TFA in precipitation reached 0.29 μg/L in the USA [11], 0.21 μg/L in Germany [1], and 0.70 μg/L in China [12]. Drinking water median TFA concentrations, ranging from 0.08 μg/L (USA) to 0.5 μg/L (Switzerland), and even up to 1.5 μg/L (Germany), were either similar to or higher than the proposed limits of total PFAS in drinking water (0.5 μg/L) in the EU Drinking Water Directive [9,13]. The thresholds for irreversible effects are unclear, but mammalian toxicity studies suggest TFA is harmful to the liver and reproduction as well [3].

As conventional water treatment technologies offer little to no removal efficiency for TFA, the risk of its accumulation is growing [3]. Consequently, environmental and health agencies are calling for tighter regulatory measures—such as restricting PFAScontaining pesticides and fluorinated gases—to limit further TFA formation [3,8,9]. In 2020, the German Federal Environment Agency (UBA) set a health guideline value of 60 μg/L for TFA in drinking water, but recommends not exceeding 10 μg/L as a precaution [14]. Other countries have set similar limits, such as 9 μg/L in Denmark and 2.2 μg/L in the Netherlands [15,16]. Recognizing the necessity for dependable monitoring, the German standardization organization DIN developed a standard for TFA analysis [7]. This method uses direct injection—liquid chromatography with tandem mass spectrometry (LC–MS/MS)—to quantify TFA in the 0.1–3.0 μg/L range and was validated by 12 labs in Germany and Switzerland [8].

Unlike most standards, DIN 38407-53 permits the use of various LC techniques, such as HPLC with HILIC, mixed-mode, and reversed-phase (RP) columns, as well as ion chromatographic approaches [7,8]. Metrohm enhanced and tailored the method for ICMS/MS analysis to meet the requirements outlined in DIN 38407-53 [7,8]. This Application Note describes the method setup using a Metrohm ion chromatograph, a Metrosep A Supp 17 separation column, and mass detection with a 6475 triple quadrupole mass spectrometer from Agilent (m/z 113 and 69). The method passed all requirements and was successfully validated during the interlaboratory trial.

EXPERIMENTAL 

Standards and samples were directly injected without further preparation steps (Figure 2). After separation on an anion-exchange column, TFA was detected with a 6475 triple quadrupole mass spectrometer from Agilent. Sample and standard handling were performed using the fast 889 IC Sample Center – cool, increasing the stability of the liquids to be measured. A full-loop injection of 100 μL was executed. All measurements, including standards, blanks (0.1% methanol in ultrapure water), and samples, were performed in triplicate. 

The separation of TFA from other anionic species was achieved on a Metrosep A Supp 17 – 150/4.0 column equipped with a Metrosep RP2 Guard/3.5 column to further improve the separation from anionic organic compounds. Isocratic conditions were used along with a carbonate eluent fortified with methanol (i.e., 5 mmol/L Na2 CO3 , 0.2 mmol/L NaHCO3 , and 10% methanol) to improve evaporation during electrospray ionization (ESI).

The IC was controlled with MagIC Net software which enables the detection of major anions, in addition to the target trifluoroacetic acid, through conductivity measurements. The MS signal was recorded using an Agilent 6475 TQ MS with a fragmentation voltage of 65.0 V to separate the ions at m/z 113 and 69. Data acquisition was performed using the Agilent MassHunter Workstation (LC/TQ) software. To synchronize the two devices, a remote box is mandatory.

CONCLUSION

The development and validation of DIN 38407-53 by IC-MS/MS marks a significant advancement in analytical methods for IC coupled with tandem mass spectrometry (MS/MS). Notably, IC-MS/MS exhibits distinct advantages when analyzing short-chain fluorinated compounds, such as TFA, due to its high matrix resistance and long column lifetimes. This makes IC-MS/MS a validated alternative approach to traditional HPLC methods. The developed method meets the requirements of DIN 38407-53. Automated features such as inline dilution further enhance the practicality of using IC for trifluoroacetic acid analysis.

3. Shimadzu: Measurement of Experimental Polar Surface Area Using Supercritical Fluid Chromatography

• Application note
• Full PDF for download

In new drug development, polarity of a drug is an important factor affecting its permeability to target sites. Polar surface area (PSA) represents the total exposed areas of polar parts on a surface of a molecule. Experimental data have demonstrated a strong positive correlation between PSA and membrane permeability^{1), 2)}. Previous studies suggested that molecules with PSA more than 140 Ų tend to exhibit poor cell membrane permeability and oral bioavailability³⁾. Molecules that can cross the blood-brain barrier are generally required to have PSA of approximately 60–70 Ų ⁴⁾. Furthermore, it has been verified that peptides with PSA more than 100 Ų do not show significant passive permeability⁵⁾. 

PSA calculated based on the bonding patterns of atoms within a molecule is called topological polarized surface area (TPSA), which is used for predicting the physical properties of candidate compounds in the early stages of drug discovery. However, TPSA is calculated without considering the three-dimensional structure of the molecule. Therefore, TPSA may not accurately evaluate the total exposed polar surface area of the molecule where its steric structure changes due to intramolecular hydrogen bonding like a peptide, a middle-size molecule. On the other hand, EPSA is a measured value based on SFC retention time (Fig. 1). Specifically, it is measured using a calibration curve created under analytical conditions where longer SFC retention times correlate with larger exposed polar area on the molecular surface. EPSA is considered effective even for molecular groups where accurate membrane permeability prediction is difficult using TPSA ^{2), 5)}. In this paper, we present a case study in which we measured the EPSA of peptides using the Nexera UC supercritical fluid chromatograph, a photodiode array (PDA) detector, and the LCMS-2050 single quadrupole mass spectrometer. Furthermore, to measure the EPSA of large numbers of compounds synthesized during the drug discovery phase with high throughput, it is necessary to maintain the instrument in a state ready for analysis at all times and to establish an environment where reliable data can be obtained regardless of how the instrument was used by the previous operator. In the latter half of this paper, we introduce a workflow for high-efficiency EPSA measurement and the implementation of open accessto the instrument using the open access software Open Solution.

Analysis of Standard Solution and Creation of Calibration Curve

The nine compounds listed in Table 1 were dissolved in dimethyl sulfoxide (DMSO) to prepare standard solution for calibration curve (each compound concentration: 1.0 g/L). These were analyzed under the conditions shown in Table 2. Fig. 3 and Fig.4 show the chromatograms of standard solution obtained using PDA detector and single quadrupole mass spectrometer LCMS2050.

Analytical Workflow Using Open Solution

Samplesin the early stages of drug discovery may contain various impurities in addition to the expected target compound. Combination use of PDA detector and single quadrupole mass spectrometer LCMS-2050 provides application of acquired spectral and mass information to identify known compounds and estimating unknown impurities as well as utilization of chromatographic retention times from PDA and MS detectors to calculate EPSA. 

Furthermore, introducing Open Solution unifies the operational workflow from sample setting sample to starting analysis to reduce human error and to improve instrument operating rate simultaneously. EPSA calculation workflow utilizing Open Solution is as follows. Analysis registration in Open Solution is designed to be completed on a single screen.

Simply select a previously registered method file from the pulldown menu, enter the targeted mass number and sample information, then click the Start key to start analysis (Fig. 12). This screen is clearly organized for easy setting of required information to allow intuitive operation even for first-time users.

During data processing phase, Open Solution Results display screen of Open Solution can be flexibly rearranged to suit user’s requirement. Consequently, UV/MS chromatograms and spectra can be organized for easy viewing to allow quick confirmation of target peak retention time and spectral information. Additionally, automatic execution of column conditioning (pre-run/post-run) for each analysis can be scheduled, resulting in optimal starting condition regardless of previous users' condition. Therefore, the necessary reliability of data required for EPSA calculation can be guaranteed. Fig. 13 shows the Open Solution results analysis screen.

Conclusion

This article describes an experimental method for measuring EPSA by combination use of SFC and single quadrupole mass spectrometer LCMS-2050, and the setup of open access environment using Open Solution to support its practical operation. EPSA calculated based on retention time in SFC will be a useful indicator for evaluating permeability, particularly for cyclic peptides and middle molecules, as it can reflect effects such as steric conformation and intramolecular hydrogen bonding that TPSA cannot fully utilize. On the other hand, in daily operations, a large number of smallvolume samples can be processed at high throughput while keeping consistent data quality utilizing functions of setup of the open access environment, automatic conditioning, and monitoring instrument condition via SST, all provided by Open Solution. This is expected to accelerate the screening and candidate selection processes in drug discovery and mediummolecule research.

4. Thermo Fisher Scientific: Enhanced analytical flexibility: simultaneous normal and reversed phase chromatography

• Application note
• Full PDF for download

This application note demonstrates how the Thermo Scientific Vanquish Flex Duo UHPLC System can simultaneously perform normal phase (NP) and reversed phase (RP) chromatography within a single instrument for the analysis of fat-soluble and water-soluble vitamins in pregnancy supplements.

Goal

The study aimed to combine NP and RP chromatography on one Vanquish Flex Duo UHPLC System to enable efficient and accurate analysis of both fat-soluble vitamin D3 and water-soluble B vitamins according to USP monographs.

Introduction

The Vanquish Flex Duo UHPLC System equipped with the NP kit enables simultaneous operation of RP and NP workflows on a single platform without requiring revalidation of USP methods. The approach was applied to pregnancy multivitamin capsules containing vitamins needed during pregnancy and breastfeeding.

The workflow included:

• NP analysis of vitamin D3
• RP analysis of vitamins B1, B2, B3, and B6
• Parallel operation on two independent flow paths

Experimental Setup

The analytical setup consisted of a Thermo Scientific Vanquish Flex Duo UHPLC System configured with:

• Dual pumps
• Dual split sampler
• Column compartment
• Variable wavelength detector
• Diode array detector
• NP compatibility kit

Chromeleon CDS 7.3.2 software was used for data acquisition and processing.

Results and Discussion

System Performance

The Vanquish Flex Duo system uses two independent flow paths with separate pumps, injectors, columns, and detectors, enabling simultaneous NP and RP operation while minimizing cross-contamination.

Special attention was given to NP column equilibration, which is critical for stable chromatographic performance.

Vitamin D3 Analysis

Six consecutive injections of the vitamin D3 standard showed excellent repeatability:

• Average peak area: 17.54 mAU·min
• Peak area RSD: 0.13%
• Peak asymmetry: 0.98

The system suitability test produced a resolution of 12.8 between cholecalciferol and precholecalciferol, exceeding the USP requirement of Rs ≥10.

Analysis of the pregnancy supplement capsules measured:

• Expected vitamin D3 content: 10 mg
• Measured amount: 9.67 ± 0.01 mg

Water-Soluble Vitamin Analysis

The RP workflow demonstrated excellent precision for vitamins B1, B2, B3, and B6. Six replicate injections produced peak area RSD values below 0.16%, well within USP limits.

Quantification results showed strong agreement with label claims:

• Niacinamide: 103.8%
• Pyridoxine: 96.0%
• Riboflavin: 112.5%
• Thiamine: 100.0%

All measured values met USP acceptance criteria of 90–125% of labeled content.

Conclusion

The application demonstrated that the Vanquish Flex Duo UHPLC System can simultaneously perform NP and RP chromatography on a single platform for comprehensive vitamin analysis. The system provided:

• High analytical flexibility
• Parallel NP and RP separations
• Excellent precision and reproducibility
• Full USP compliance
• Accurate quantification of fat-soluble and water-soluble vitamins

The workflow significantly improved efficiency and throughput while maintaining robust chromatographic performance and reliable quantitative results.

5. Waters Corporation: Automating Sample Extraction and Cleanup of Per- and Polyfluoroalkyl Substances (PFAS) in Fish Tissues Following EPA 1633 Guidance

• Application note
• Full PDF for download

Benefits

• A fully automated comprehensive workflow including sample extraction and clean-up for PFAS analysis of fish tissues produces high quality data with reduced manual efforts, while maintaining compliance with the EPA 1633 procedure. 
• Performance criteria of EPA 1633 for extraction and analysis of tissue samples were easily met using a fully automated workflow, delivering accurate results with greater efficiency and confidence. 
• An automated sample preparation workflow optimizes laboratory efficiency by reducing sample preparation time from 2 days to a single 8-hour shift minimizing overall time to process samples.

US EPA Method 1633 is a multi-lab validated method for the targeted analysis of 40 PFAS in non-potable water matrices, soils, biosolids and tissues.¹ For tissue samples, the sample is first extracted followed by clean-up using graphitized carbon black (GCB) and a weak anion exchange (WAX) SPE cartridge. The initial sample extraction method is a lengthy 3 step protocol where the first step requires a 16-hour extraction. If this method is performed full manually, it is a 2-day process to complete the extraction, sample cleanup and analysis which impacts sample throughput and turn-around time. Automation of the sample preparation workflow can significantly decrease the sample processing time and ease the burden of challenging methods on laboratory staff. 

In previous work, the manual workflow for tissue analysis following EPA 1633 was presented and tested.² The current work adapts the use of automated sample preparation systems; a pressurized fluid extraction system (EDGE PFAS™ System from CEM) and an automated SPE extraction system (SPE-03 Gen 4 from PromoChrom). Using both systems to automate the sample preparation and clean-up reduced the 2-day process to one that is achievable in a single 8-hour shift with minimal human interaction. Utilizing an automation workflow enhances the already reliable solution of the ACQUITY™ Premier System coupled to a Xevo™ TQ Absolute Mass Spectrometer for PFAS analysis following EPA Method 1633.

Experimental

• LC system: ACQUITY Premier System with Binary Solvent Manager and Flow Through Needle
• Analytical column: ACQUITY Premier BEH™ C18 Column 2.1 x 50 mm, 1.7 µm
• Isolator column: Atlantis™ Premier BEH C18 AX Column 2.1 x 50 mm, 5.0 µm
• MS system: Xevo TQ Absolute Mass Spectrometer
• Software: waters_connect™ for Quantitation Software

Conclusion 

Sample preparation for fish tissues following the EPA 1633 guidance was successfully automated using the CEM EDGE PFAS and PromoChrom SPE-03 systems. The overnight extraction step required when performing this method manually was reduced to about 10 minutes per sample, allowing a batch of 12 to process in approximately 2 hours. Additionally, the SPE clean-up process was also fully automated requiring only a 2-hour hands-free method. The combined procedures using both automated systems allows for the reduction of sample preparation from a 2 day process to one that is easily achievable in an 8 hour shift. 

It was shown that neither automated system contributed any PFAS contamination to the samples and is therefore suitable for accurate and confident PFAS analysis even at trace levels. Extracted internal standard and fortified native PFAS recoveries in salmon, tuna and shrimp were well above the required minimum recovery values and shown to be equivalent to the manual process previously evaluated. Additionally, calculated concentration values for a fish reference material closely matched expected results, reinforcing confidence in method accuracy. The data demonstrates that using automated pressurized liquid extraction in series with automated SPE extraction is equivalent to processing tissue samples manually, giving laboratories more flexibility in sample handling while increasing sample capacity for EPA 1633.