The Power of Precision
ZenoTOF 7600 system
TripleTOF 6600+ system
SCIEX 7500 system
SCIEX Triple Quad 6500+ system
SCIEX 5500+ system
QTRAP 6500+ system
QTRAP 4500 system
View all mass
Echo MS system
2.0 High Throughput Multiplexing system
View all integrated
BioPhase 8800 system
8000 Plus system
800 Plus system
Advance your research with front-end
instruments designed to help you realize the full power of your mass
spectrometer. SCIEX has the broadest portfolio of ESI-MS front-ends
can facilitate various flow rates, sample requirements and
sensitivities. No one else offers the entire range
flow, microflow, nanoflow LC-MS and even ultra-low flow
ExionLC 2.0 series
View all front-end HPLC MS
Differential mobility spectrometry (DMS) and
ion mobility spectrometry are analytical techniques used to separate
ions based on their gas phase mobility. Multiple types of ion
devices exist, such as drift tubes, traveling wave, and high-field
asymmetric waveform devices. Learn how you can separate yourself
Differential Ion Mobility.
The latest ion sources from SCIEX enable
enhanced sensitivity and robustness with greater desolvation range
across all MS platforms, from Triple Quad to QTRAP and QTOF.
Turbo V ion
OptiFlow Turbo V ion
View all ion sources
vMethod applications are pre-configured and verified LC-MS/MS
that reduce the need for method development – significantly cutting
time, effort and money to deploy a new assay. Every vMethod provides
method conditions, recommended sample prep, LC and MS conditions,
details for applicable MS/MS library databases for key applications.
and personal care products (PPCPs)
and protein bioanalysis
Explore vMethod applications
Free software trials
feature request portal
Analyst TF software
Biologics explorer software
Molecule profiler software
High resolution and QTRAP
libraries can dramatically enhance the quality of your analysis,
you much improved confidence in your data. With a comprehensive
at your fingertips, you can easily create methods and process
and non-targeted screening data on your complex samples, faster and
easier than ever before.
All in one
HR-MS/MS library with NIST 2017
Explore the library
Boost the performance of your mass
spectrometer and improve sensitivity, productivity, and data
iChemistry Solutions are the world's only reagents and
that are custom designed with your success in mind.
RNA 9000 Purity & Integrity kit
kit for amino acid analysis of hydrolysates
kit for amino acid analysis of physiological fluids
protein digestion solution
Biologics analysis kit for
protein assay - human induction kit
CZE rapid charge variant analysis kit
glycan analysis and labeling for the PA 800 plus
standards kit for pesticide analysis
auEChERS extraction and dispersive kit
Ampliflex keto reagent
Cleavable ICAT reagent
View all consumables
QTOF – Quadrupole
Time of Flight
QTRAP® – Triple Quad Linear
SWATH® – Data Independent
SelexION® – Differential
MicroLC – Microflow
View All Technology
From substantiating new discoveries through
to end-to-end validated workflows for high-throughput biomarker
in the clinic, thousands of scientists worldwide depend upon mass
innovation from SCIEX to advance their work in the fields of
research, omics and diagnostics.
View All Clinical
Pesticides & herbicides
Soil and biota
View All Environmental
How do you protect your reputation and meet today’s global food
standards? Whether you are a commercial lab or a food manufacturer,
quality of your food testing data is vital to your business. SCIEX
solutions help you meet maximum residue limits (MRLs) with
data that you can genuinely count upon. With a portfolio of
applications, your lab can quickly and easily react to diverse
Authenticity & Profiling Analysis
& Food Contact Substance Analysis
Food and Beverage Testing
How do you ensure the integrity of your
results in an industry that is never constant? By accurately
even the smallest compound angles you can deliver evidence that
SCIEX forensic analysis solutions deliver fast, highly accurate
data across a multitude of compounds and biomarkers, from the known
the new and novel.
View All Forensics
and SNP Analysis
View All Life
Transform the capacity and capability of your biologics pipeline
complete end-to-end solutions that make your lab more productive,
more successful. With a longstanding track record in pharma
development and manufacturing, our unparalleled application
with best-in-class hardware, software and support all integrate to
revolutionize your lab.
Native Mode Analysis
Nucleic Acid and Plasmid Analysis
View All Pharma
Mass Spec Operators
View All Diagnostics
The SCIEX Now Learning Hub offers the most diverse and flexible learning options available, with best-inclass content that helps you to get the most out of your instrument and take your lab to the next level. Available personalized learning paths based on the latest memory science ensure better knowledge retention, and automated onboarding and enrollment means you’ll get up and running faster.
SCIEX Now Learning Hub offers the most diverse and
flexible learning options available, with best-in-class content that
helps you to get the most out of your instrument and take your lab
the next level. Available personalized learning paths based on the
latest memory science ensure better knowledge retention, and
onboarding and enrollment means you’ll get up and running faster.
SCIEX Learning Manager provides you with the infrastructure to assign, monitor and report on your staff's
competency through a single digital platform. Effectively manage the training process for new hires, ensure
continuous staff development and access information with a single log-in to your SCIEX account.
You can browse, filter, or search our
extensive list of training offerings. Choose from over 100
eLearnings or search for an instructor-led course near you. Once you
select the course you want to take, you will be directed to
Learning Hub for enrollment (login required).
SCIEX Now Online is the Everything, Anytime
destination for all your SCIEX support needs. You can keep track of
activities that matter most to you and manage your lab in the most
efficient way possible. Extensive self-help resources like our deep
Knowledge Base, enable you to solve many problems on your own. SCIEX
is available 24/7 and your new SCIEX instruments are automatically
to your profile when you purchase.
SCIEX Now Learning Hub
Manage My Instruments
SCIEX Now New Feature Request
Software New Feature Request
Online ordering solutions
Don't have an
account? Create One
Visit your SCIEX
No one understands your needs and can support
your systems better than we can. Our mission is to help you be
successful, whether it's to repair your instrument, assist with your
workflows, or help you maximize productivity in your lab. Whatever
challenge, global SCIEX Service and Support personnel are subject
experts who are focused on mass spec and capillary electrophoresis,
you'll be able to achieve your scientific goals quickly and
Plus Suite for Your New LC-MS
Instrument Service & Support
View All Support Tools
SCIEX Now Learning Hub is much more than online
courses. For the most comprehensive option, you can select Success
Programs: personalized, blended online and in-person courses. If
needs are met by a visit from one of our training experts, you can
choose multiple Onsite Training days to get your lab running. Visit
SCIEX training center in North America, Europe or Asia for intensive
classroom and laboratory training. And your online courses are
any time, from anywhere, right here.
Login to SCIEX Now Learning Hub
Success Programs at Your
Scientist Training at Your Site
Visit all Training
If you have CE, LC or mass spec questions,
then SCIEX has the answers. SCIEX support is the single destination
your system questions. We aim to fully assist you with virtually
application of our instruments, helping you to get the most out of
lab resources and assets.
View All Request Support
As a life science researcher, you need the
tools and support to help you create the scientific foundation in
pursuit of expanding the knowledge-base, whether it is understanding
fundamental biology, finding new biomarkers, discovering ways to
our quality of life, or other areas of research. We are committed to
same goals and put the very same dedication into our work to help
address your most significant research challenges.
View All Partnership
Regulatory compliance is as paramount for us
as it is for you. That’s why we have made it easy for you to freely
reference all relevant technical and product regulatory documents.
give you confidence that, with SCIEX, you will fully comply with
legislation, adhere to your laboratory protocol and meet industry
Declaration of Conformance
Safety Data Sheets
Certificates of Analysis
View All Regulatory Documents
SCIEX supplies extensive documentation to help you prepare for, use, and maintain your SCIEX hardware and software products, and we update this documentation regularly. On the Customer Documents page, you can search for and download the latest documents for your product.
Join the SCIEX community today to
interact with your peers, share and exchange ideas, develop
your knowledge, stay up-to-date with the latest products,
post insights and questions, comment on others and receive
support. This community is designed to help you, our
customers, move science forward and get the answers you
need. We’re committed to engaging with and listening to you,
to create the best customer experience possible and to
contribute to the success of your work.
Environmental / Industrial
Food and Beverage
Life Science Research
Now Feature Requests
View All Community
Our favorite papers
Meet our executives
In the news
You've got questions. We've got experts who
can help. Contact us to find out more, talk to a specialist, explore
solutions or get expert support.
Request a quote
SCIEX success network
Request hosted catalog
Global public relations
Bosnia and Herzegovina
United Arab Emirates
United Republic of Tanzania
AB Sciex is doing business as SCIEX. © 2010-2018 AB Sciex. The trademarks mentioned herein are the property
of the AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license. Beckman
Coulter® is being used under license. Product(s) may not be available in all countries. For
information on availability, please contact your local representative. For
research use only. Not for use in diagnostic procedures.
Download tech note (PDF)
Using Scheduled MRM™ Algorithm on the SCIEX QTRAP® 6500+ LC-MS/MS System
Pierre Negri1 and Scott Krepich1
1SCIEX, Redwood City, CA, USA
Here, a comprehensive workflow using the SCIEX QTRAP 6500+ System for the analysis of 22 PFASs in serum is presented. This targeted screening workflow is shown to provide a fast, robust and sensitive analytical method capable of accurately quantifying sub-nanogram per mL levels of PFASs in human serum.
Per- and polyfluoroalkyl substances (PFAS) are pervasive compounds used in a variety of industrial applications and found in a wide range of consumer products such as cookware, stain repellant, flame-retardant and coatings.1 PFAS are considered environmental factors due to their persistent and bioaccumulating nature. A recent biomonitoring study conducted by the National Health and Nutrition Examination Survey (NHANES) on a nationally representative sample of the U.S. population found that more than 98% of the people tested had multiple congeners of PFAS present in their bodies.2
Bioaccumulation of PFAS in the human body resulting from environmental exposure is a growing public health concern. Recent studies have linked PFAS exposure to adverse health outcomes including childhood health complications, reduction in kidney functions, thyroid disease, hormone suppression, decreased fertility, increased cholesterol levels and diabetes, among others.3,4,5,6 Given the prevalence and ubiquitous nature of PFAS in the environment and every-day consumer products (including our drinking water supply), there is a critical need to develop quantitative tools capable of accurately and precisely detecting low-levels of PFAS in biological fluids to inform the extent of their bioaccumulation and overall impact on the human body. Close biomonitoring of PFAS levels will help evaluate their toxicity and further understand the health consequences associated with their bioaccumulation over time in exposed human populations.
In this technical note, a quantitaitive workflow for the analysis of 22 PFAS in serum was developed using the SCIEX QTRAP 6500+ System. This targeted screening workflow provides a fast analytical method capable of accurately quantifying sub-nanogram per mL levels of PFAS in the human body.
Figure 1: Accurate and sensitive detection of PFAS extracted from serum samples. Representative extracted ion chromatograms (XICs) of the qualifier ion for PFHxS, one of the most persistent PFAS measured at the highest levels in exposed human population. From left to right: in matrix blank, at 0.1 ng/mL (LOD) and 0.5 ng/mL (LLOQ).
Standards and internal standards: A total of 22 PFAS and 15 mass-labeled internal standards were purchased from Wellington Laboratories (Guelph, ON, Canada) and prepared using Baker’s HPLC-grade methanol. A list of all the PFAS included in this panel is summarized in Table 1.
Calibrator preparation: A 1 µg/mL stock standard solution mixture containing the 22 PFAS was prepared by diluting the stock standard solutions with methanol. The resulting 1 µg/mL stock standard solution mixture was used to spike 50 µL of serum in order to prepare a series of 9 calibrator solutions covering concentations ranging from 0.01 to 100 ng/mL. A 1 µg/mL stock internal standard solution mixture containing the mass-labeled internal standards was prepared similarly by diluting the stock standard solutions with methanol. This solution mixture was used to prepared a 5 ng/mL mass-labeled internal standard solution in 0.1 M formic acid. High density polyethylene or polypropylene consumables (Eppendorf tubes, pipette tips, HPLC vials, etc) were used to minimize PFAS contamination from external sources.
Sample preparation: PFAS were extracted from 50 µL serum samples by using a protein precipitation procedure summarized in Figure 2.
Figure 2. Protein precipitation procedure for serum samples. A 10-step protein precipitation procedure was used for extracting the PFAS from serum samples for MS analysis.
Liquid chromatography: UHPLC separation was performed on a Phenomenex Gemini® C18 column (50 x 2 mm, 3 µm, 00B-4439-B0) at 25ºC on a SCIEX ExionLC™ AC System. A Phenomenex Luna C18(2) column (30 x 2 mm, 5 µm, 00A-4252-Y0) was installed between the pump mixing chamber and the analytical column used for separation. This additional column served as a delay column to isolate PFAS contamination leaching from the LC system components and minimize the risk of system-related PFAS interfering with real signals from the sample during the analytical run.
In addition, the fluoroethylene and Teflon tubing on the ExionLC AC System pumps and degasser was replaced with PEEK tubing to minimized the impact of PFAS background contamination and leaching. The FEB and PTFE tubings from the rinse solvent lines, the needle seal, the sample holding loop, pump seal, pump lining and degasser unit were also replaced with PEEK tubing to avoid system-related interferences. Additionally, the PTFE frits and rotor seals were replaced with stainless-steel parts. Mobile phases used for analytical separation were ammonium acetate in water (mobile phase A) and formic acid in methanol (mobile phase B) prepared from HPLC and LC-MS/MS grade solvents. The LC flow rate was 0.6 mL/min and the total run time was 6.5 min. The injection volume was 10 µL. Each extracted sample was injected in triplicate over the course of two consecutive days.
Mass spectrometry: Data were collected using a SCIEX QTRAP 6500+ System with a IonDrive™ Turbo V Ion Source, operated with electrospray ionization (ESI) in negative mode. The ion source parameters were optimized for the LC conditions using the compound optimization (FIA) feature in Analyst® Software. A single acquisition method consisting of 57 MRM transitions (42 for the PFAS and 15 for the mass-labeled internal standards) was created in Analyst Software. The Scheduled MRM Algorithm was used to optimize data sampling across each peak and maximize the dwell times used, ensuring reliable integration, quantification and confirmation for each of the PFAS in the panel.
Data analysis: Data processing was performed using SCIEX OS Software. Detection and integration of the peaks from the background was achieved using the MQ4 algorithm. Quantitative analysis was performed in the Analytics module of the software where calibration curves, concentration calculations, assay precision and accuracy statistics were automatically generated.
PFAS are pervasive and persistent compounds that have the potential to accumulate and contaminate the LC systems used in analytical testing. As a result, measures were implemented to reduce the risk of outside, ambient and system-related PFAS contamination. The most critical measure was the inclusion of a delay column between the autosampler and LC pumps to trap ambient and system-related PFAS and ensure they will be retained away from the sample signals.
Figure 3 summarizes the chromatographic response following the addition of the delay column and shows the extracted ion chromatograms (XICs) for PFHpA (top row) and PFHxS (bottom row) before and after the hardware modifications were made on the LC system.
Figure 3A shows the background signal resulting from a blank sample injection before the system modifications were made. Sharp PFAS peaks resulting from the ambient LC system contamination were observed throughout the chromatograms, including at the retention times at which the sample peaks were expected.
Figure 3B shows the background signal of the same blank sample injection after the system modifications were made (including the addition of the delay column). This configuration eliminated the PFAS interference peaks at the expected analyte retention times and produced a much broader and delayed contaminant peak caused by the system-related PFAS that were held up by the delay column.
Figure 3C shows the XICs resulting from a 10 ng/mL injection of a sample containing all the PFAS in the panel with the system modification. The XICs show a sharp peak resulting from the PFAS in the samples followed by the same broad and delayed contamination peak shown in Figure 3B. The addition of the delay column and the modifications made to the LC system components together minimized the impact of system-related PFAS contamination and ensured the analytical integrity of this quantitative workflow.
Figure 3. Benefits of using a delay column for PFAS analysis. XICs for PFHpA (top row) and PFHxS (bottom) showing (A) background and system-related contamination signals resulting from a blank sample injection before the system modifications were made showing, (B) broader and delayed contaminant peaks caused by the system-related PFAS that were held up following the addition of the delay column, and (C) sharp peaks resulting from the PFAS in the samples followed by the delayed and broad contamination peaks caused by the delayed column. The modifications made to the LC components significantly reduced the impact of system-related PFAS interferences and enabled accurate quantification of PFAS in serum samples.
Chromatographic separation of PFAS, including the two compounds that contain both branched and linear isotopes (PFOS and PFHxS), is critical to ensure reliable and accurate quantification. Figure 4 shows the chromatographic profile on an injection of the neat, 10 ng/mL standard solution containing the 22 PFAS. The choice of column, gradient and optimized mobile phase composition resulted in the baseline separation that was needed to correctly distinguish all isomers. As seen in Figure 4, the delayed contamination peaks caused by the delayed column did not interfere with the PFAS sample peaks.
Figure 4. Chromatographic profile of the 22 PFAS monitored in this study. Extracted ion chromatograms (XICs) resulting from the optimized data acquisition method using a 10 ng/mL neat standard mixture. The combination of the optimized mobile phase composition and column choice enabled baseline separation of the PFAS congeners. This method has also shown the ability to separate branched and linear isomers of individual PFAS chain lengths (not shown here).7
Blank serum samples were spiked with the standard mixture containing the 22 PFAS at concentrations ranging from 0.01 to 100 ng/mL. These standard solutions were extracted using the protein precipitation procedure and injected in triplicate. Data were processed in the Analytics module in SCIEX OS Software 2.0 using the MQ4 Algorithm.
Calibration curves were generated for each of the PFAS in the panel and plotted across 9 calibration levels ranging from 0.1 to 100 ng/mL to evaluate the linearity of the method for the serum-spiked control samples. Figure 5 shows the resulting calibration curves for the native linear perfluoroalkylsulfonates (A) and the native linear perfluoroalkyl carboxylic acids (B). These calibration curves demonstrated excellent linearity covering 3 orders of magnitude with R2 greater than 0.99 for all of the PFAS in the panel with the exepction of PFODA. The system modifications implemented in this method were critical in attaining linearity achieved in this workflow.
Figure 5. Excellent linearity for the 22 PFAS. Regression curves resulting from the calibration series from 0.5 to 100 ng/mL for A) native linear perfluoroalkylsulfonates and B) native linear perfluoroalkyl carboxylic acid extracted from serum samples. R2 values greater than 0.99 were observed for all of the PFAS in the panel.
The ability to accurately detect low levels of PFAS extracted from serum samples is critical to inform the extent of their accumulation in the human body. The series of calibrators was injected to evaluate the ability to quantify PFAS across a wide range of concentrations. Figure 6 shows representative extracted ion chromatograms (XIC) for the two MRM transitions monitored for PFHxS and PFOA, two PFAS commonly measured in the exposed population. Five levels of calibrators were used to determine the ion ratio criteria for the quantifier and qualifier ions of PFHxS and PFOA. The series of XIC traces for the two compounds showed a high level of consistency and precision, as evidenced by the acceptance criteria (20% or less) of all the ion ratios across the calibration series ranging from 0.5 (LLOQ) to 100 ng/mL. However, the majority of the PFAS showed detectable signal below the LLOQ. Figure 1 shows the XICs for PFHxS for the matrix blank (left), at 0.1 ng/mL (middle) and 0.5 ng/mL (right). The signal at 0.1 ng/mL is the limit of detection (LOD) for PFHxS as it is well above the blank signal. Similar peaks were observed below the LLOQ for the PFAS in the panel. In addition, the assay showed great reproducibility over the course of the two consecutive days the data were acquired (inter-day peak area variations of 5% or less were observed for the 22 PFAS across the calibration range).
The lower limit of quantification (LLOQ) for each of the PFAS included in the panel was determined based on the lowest concentration at which the integrated peak area of the analyte was quantifiable, with calculated concentration accuracy between 80 and 120%, precision (%bias) below 20%, ion ratio acceptance criteria of less than 20% and maintaining a linear calibration curve with an R2 value of at least 0.99.
Figure 6. Accurate quantification of bioaccumulating PFAS in serum. Extracted ion chromatograms (XICs) for A) PFHxS and B) PFOA, two of the most persistent and common PFAS measured in the exposed human population. The XICs display overlays of both the quantifier and qualifier ions from 0.5 to 100 ng/mL as well as the tolerance in the form of the ion ratio line overlay that help in visualizing the confidence levels for the ion ratios. All ion ratios passed the ion ratio acceptance criteria (20% or less) across the calibration range (0.5 to 100 ng/mL).
Table 1 summarizes the quantitative performance of the workflow and includes the calibration range, linear correlation coefficient (R2 value), LLOQ, as well as the accuracy and precision values at the LLOQ for each of the 22 PFAS monitored in this workflow. PFBS and PFODA are the only two PFAS in the panel that proved challenging to quantify accurately due to their ubiquitous presence in the analytical system. This was evidenced by their detection in blank and solvent injections and as a result, their pervasiveness impacted their detection limits in this workflow. PFBS was quantified accurately from 5 to 100 ng/mL however PFODA could not be quantified.
The assay as a whole demonstrated excellent reproducibility, linearity, precision and accuracy for all the other PFAS in the panel. This method achieved the required levels of robustness and qualitative performance necessary to accurately measure sub ng/mL levels of PFAS from serum samples.
Table 1. Statistical results for the 22 PFAS monitored in this workflow. The table includes calibration range, linear correlation coefficient (R2 Value), and LLOQ, as well as the accuracy and precision at the LLOQ for each of the PFAS MRM transitions monitored.
A robust and sensitive workflow for the detection of PFAS in serum samples using the SCIEX QTRAP 6500+ System was successfully developed. The addition of a delay column and the modifications made to the LC system components reduced the risk of system-related PFA interferences. The combination of a simple sample preparation procedure with a fast LC separation enabled accurate and sensitive detection of 22 PFAS down to sub ng/mL levels. The assay showed excellent reproducibility, precision, accuracy and linearity, with an LLOQ of 0.5 ng/mL, LOD of 0.1 ng/mL and an R2 of greater than 0.99 for the vast majority of the PFAS in the panel with the exception of PFBS and PFODA. The excellent precision and accuracy observed at the LLOQ is highlighted in Table 1.
Overall, the developed method provides a robust and accurate method for bio-monitoring of low-levels of PFAS in biological fluids. Therefore, the presented workflow is readily adaptable for high-throughput toxicology investigations aimed at determining the extent of PFAS bio-accumulation and its broader impact on human health.
The Destination for All Your Support Needs