Break the boundaries: How to perform reliable quantitative LC-MS measurements with maximized sensitivity

LC-MS innovation: Improve your analytical accuracy and maximize sensitivity

Liquid chromatography–mass spectrometry (LC-MS) is a powerful analytical technique commonly used for separation, identification, and quantification of both known and unknown compounds. LC-MS is widely recognized as a specialized tool that generates highly accurate results with enhanced sensitivity and precision, as well as a large dynamic range of detection. This technique is becoming the preferred tool to use by many liquid chromatographers, since it combines the physical separation capabilities of liquid chromatography, along with the detection specificity of mass spectrometry – which creates and also detects charged ions. LC-MS now means that the analysis of complex biological samples that once were difficult to analyze have become a thing of the past.

Selecting an appropriate LC-MS solution to meet your needs can be a challenge. Users need to consider sample type and application, and find out how to best optimize their sample preparation processes. The level of LC-MS performance must be suitable to meet user needs, while also considering particle and stationary phases, LC accessories, and reference materials. There are many ways to identify whether you need to consider buying a new LC-MS instrument, and this choice should be application driven. For instance – if an instrument has not been refurbished, sensitivity and speed can be lost overtime – since internal components age, which makes it difficult to keep up with newer technologies. Plus, if an instrument is not sensitive enough to detect or quantitate the levels you require, then it is likely time to consider an upgrade. If a user needs faster speed to improve productivity or throughput, then they may need to consider an LC-MS instrument that has been introduced within the past year – since speed has been a key focus in the development of the majority of state-of-the-art LC-MS instruments.

To help overcome these issues, Restek have been working to perfect the art and science of liquid chromatography by creating a range of specialized tools designed to streamline analyses, and help users master their analytical results.

Grains and seeds

Hear from Jeanette Langner, head of the department of pesticides in food at PiCA Prüfinstitut Chemische Analytik GmbH as she explores the challenges she faced when detecting polar compounds in food matrices, and why she chose Restek Polar X LC Columns to streamline these analyses.

Read article

Assay optimization

Assay optimization can be challenging, particularly for those working within therapeutic drug monitoring(TDM). TDM is a clinical practice used to measure drugs at designated intervals to maintain a constant concentration in the bloodstream, in order to optimize individual dosage regimens and avoid patient toxicity. New and improved pain management medications are consistently being developed and released onto the market on a regular basis. The steady increase of pain medication has led to many hospital and reference labs working tirelessly to screen more patients.

Patient samples must be screened to prevent drug misuse and to ensure patient safety. The tests needed to monitor therapeutic drugs can be challenging, whereby technicians need to manage low cut-off levels, matrix interferences, and isobaric drug compounds. LC-MS/MS tools can be used to address these challenges, by delivering increased speed, sensitivity, and specificity.

The Raptor™ Biphenyl column from Restek has proven as an ideal tool for developing successful LC-MS/MS pain medication screening methodologies, since the column has been designed with high retention and unique selectivity. The column has enabled 231multiclass drug compounds and metabolites – including over 40 isobars to be analyzed within 10 minutes, as shown in Figure 1. In addition, separate panel shave also been optimized on the Raptor Biphenyl column for use with arange of specialized drugs, including opioids, antianxiety drugs, barbiturates, and analgesics – for use during confirmation and quantitative analyses.

Figure 1

Figure 1: Multiclass Drug Panel on Raptor Biphenyl by LC-MS/MS

In the key resources below, we explore pain management using the Raptor Biphenyl LC columns, and explain how these columns enable fast separation in bioanalytical testing applications, such as pain panels. We also reveal the importance of mycotoxin detection in raw plant materials and cannabis products, and present a simple pass-through solid phase extraction (SPE) sample clean-up method, which was applied to CBD oils using the Raptor Biphenyl column.

The power behind separation

LC-MS is a powerful separation technique and is also the preferred tool used to separate large non-volatile molecules, such as complex peptides and proteins. C18, along with C8 and C4 are all linear alkylsilane phases. C18 is octyldecylsilane and contains 18 carbons bound to silica. C18 analytical columns are the cheapest and most common form of column used for reverse-phase chromatography. These columns comprise of a hydrophobic surface, and a large surface area, due to their large number of carbons. These columns are available with a range of characteristics to meet the unique needs of the user, and can be used across many disciplines, from pharmaceuticals to environmental science.

Despite the popularity of C18 columns, they do not work effectively for every compound. As an alternative, users can opt to use the Raptor™ FluoroPhenyl column, a column which has been developed to deliver greater selectivity and retention for analytes that are not easily separated by C18 phase chemistry. For instance, C18 columns cannot effectively separate isobaric epimers, while the FluoroPhenyl column provides chromatographic resolution to ensure accurate results are generated.

Figure 2

Figure 2: 25-HydroxyvitaminD2/D3 and 3-Epi-25-Hydroxyvitamin D2/D3 on Raptor™ ARC-18 by LC-MS/MS

Figure 3

Figure 3: 25-HydroxyvitaminD2/D3 and 3-Epi-25-Hydroxyvitamin D2/D3 on Raptor FluoroPhenyl by LC-MS/MS

Raptor FluoroPhenyl LC Column

Figure 2 and Figure 3 present a comparison between the C18 and the Raptor FluoroPhenyl column and demonstrates their ability to separate the epimers of vitamin D2 and D3 25-hydroxymetabolites. For additional information on the analysis of epimers of 25-hydroxyvitamin D in serum, look at the resource below.


Rapid Analysis of C3 Epimers of 25-Hydroxyvitamin D in Serum by LC-MS/MS

Mastering your results

The retention and separation of challenging compounds can be laborious, time-consuming, expensive, and above all, complex. Therefore, the most appropriate technology must be selected to ensure the most accurate results are generated as simply as possible. Restek have created a range of bespoke LC-MS tools designed to help users easily master their analytical results.

In the resources below, hear from the experts at Restek as they introduce a simple approach to the retention and separation of challenging compounds, including a unique stationary phase created to selectively retain polar analytes. Plus, find out more about how to best monitor biomarkers, and learn how methods for monitoring alcohol consumption biomarkers (EtG and EtS) are limited by poor retention and coelution, and how the dilute-and-shoot EtG/EtS LC-MS/MS analysis method was established. Discover how this method was developed using the Raptor™ EtG/EtS column, designed to resolve EtG and EtS from matrix interferences, and provides consistent, accurate results for high-throughput lab testing. These resources will also explore reference standards and the key steps needed to build a solid analytical foundation.

EtG/EtS LC-MS/MS Analysis

Raptor EtG/EtS LC Column

Raptor Polar X LC Columns

To help take your analysis to the next level, discover the Force LC column, with Time-Tested Restek Biphenyl Phase. This tool has been designed to separate compounds that other phenyl and C18 chemistries cannot perform, including the use of simple, MS-friendly mobile phases. The Force LC column is particularly valuable for those working within therapeutic drug monitoring or toxicology. For instance, Figure 4 outlines how the Force Biphenyl columns were created for applications where fast, reliable identification of drugs and metabolites are necessary.

Figure 4: Morphine and Metabolites on ForceBiphenyl by UHPLC-MS/MS

Additionally, the Force LC columns also have a General-Purpose Restek C18 Phase, designed to offer high hydrophobic retention, along with a wide pH range, which delivers excellent data quality over a range of applications. This phase is compatible with moderately acidic-to-neutral mobile phases, and generates excellent data quality in food, environmental, and bioanalytical applications.

Column description

Force Biphenyl LC Column

Force C18 LC Columns

Enhance LC-MS technology workflows

In-line sample preparation (ILSP) uses LC-MS/MS to streamline and automate sample extract clean-up, which, in turn, reduces cost, sample preparation time, and also eliminates downtime between samples.

For those working within the food safety sector, the Revive ILSP Pesticides cartridges from Restek are an ideal sample preparation alternative for researchers who need to spend less time and money on multiresidue pesticide samples, without sacrificing performance. These cartridges function by effectively separating analytes from potentially interfering matrix components, like conventional SPE and QuEChERS, but it also replaces the time-consuming manual procedures via an automated clean-up process of complex samples. Figure 6 presents a comparison between the Revive ILSP Pesticides workflow and a QuEChERS workflow for the analysis of pesticide residues in spinach.

This diagram demonstrates that the ISLP method resulted in a time saving of 1.5 hours, with fewer sample transfers, and also minimized the risk of introducing error through manual steps. Figure 7 provides more information on how Revive In-Line Sample Prep operates to deliver superior analytical results.

Figure 5: An image to illustrate the Revive in-line sample preparation system – operating 1.5 hours faster than a traditional QuEChERS workflow for 14 spinach samples.

ISLP Workflow

Figure67: A diagram to demonstrate how In-Line Sample Preparation operates.

In-Line Sample Preparation

Revive In-Line Sample Preparation

Raptor EXP® Guard Column Cartridges

The technology designed to meet all your LC-MS needs

Raptor™ ARC-18 LC Columns

The Raptor™ARC-18 Column is made specifically for high-throughput, low–sample prepLC-MS/MS applications. A well-balanced retention profile offers betterdetection and integration of large, multiclass analyte lists, and stericprotection helps this SPP column endure the harsh, low-pH mobile phases neededfor mass spec without sacrificing retention or peak quality.

Raptor™ ARC-18 LC Columns
Raptor™ Biphenyl LC Column

Raptor™ Biphenyl LC Column

With Raptor™ LC columns, Restek chemists became the first to combine the speed of superficially porous particles (also known as SPP or “core-shell” particles) with the resolution of highly selective USLC® technology. This new breed of chromatographic column allows you to more easily achieve peak separation and faster analysis times without expensive UHPLC instrumentation.

*Certain images and/or photos on this page are the copyrighted property of, its contributors or its licensed partners and are being used with permission under the relevant license. These images and/or photos may not be copied or downloaded without permission from Other images courtesy of Restek..