Agilent 6495B Triple Quadrupole LC/MS System

EXPERIENCE THE HIGHEST

LEVEL OF CONFIDENCE
AGILENT 6495B TRIPLE QUADRUPOLE LC/MS SYSTEM

THE HIGHEST LEVEL OF CONFIDENCE

Your samples are precious and turnaround time is critical to the success of your organization. A sensitive, yet rugged
instrument that provides day-in, day-out performance is cornerstone to your workflow. The Agilent 6495B Triple
Quadrupole LC/MS system is at the forefront of robustness, reliability and accuracy, which makes it a perfect option for
many applications, including; peptide quantitation, food safety, environmental, clinical research and forensics.

iFunnel
Provides unprecedented
sensitivity by providing high ion
acceptance and transmission
with our proprietary technology

Gate valve
Enables higher uptime and increased
productivity by allowing capillary replacement
without having to vent the instrument

Q1 Ion optics
Reduces contamination and enables lower limits of detection
by increasing ion transmission efficiency to the Q1 mass filter

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 Ion Detector
Increases sensitivity and quantitation
across a wide mass range using a high
energy conversion dynode with low noise
characteristics

Curved Collision Cell

A tapered hexapole design with axial
acceleration, facilitates efficient collection
and transmission of fragment ions, while
eliminating cross-talk

For more information, go to www.agilent.com/chem/6495B

3
 2.46
0.0197 18.5 97.5
×10 2
0.0394 18.3 104.2
0.0098 ng

Counts
2.48
0.0591 12.5 108.3
2.46
0.0985 9.3 102.4
×10 2
0.197 13.0 95.6
0.0197 ng

Counts
2.5 0.394 5.9 84.8
0.985 5.7 102.1

ANALYTICAL CONFIDENCE
×10 2
1.97 1.6 105.5
0.0394 ng

Counts
2.6 19.7 2.9 99.7

×10 2
0.0591 ng

Counts
Targeted Proteomics 2.6

0.90 0.95 1.00 1.05 1.10

Acquisition time (min)

Peptide identification and quantification 932.7 & 204.1

20,000
18,000 932.7concerning 1,400
& 2,227.9 size
High m/z peptide ions are extremely descriptive and provide important biological information and location of post-translational
16,000 932.7 & 2,065.8 1,200
modifications (PTMs), for example glycosylation’s.
14,000 932.7 & 1,392.6
Response

Response
1,000
12,000
The Agilent 6495B Triple Quadrupole LC/MS10,000system mass range extends to m/z 3000, which 932.7is&beneficial
366.1 in 800
detecting large peptide fragments.
Here we demonstrate the utility of the 6495B8,000
for detecting fragment-ion signals arising from peptides, representing
600 each of the targeted
proteins. This approach is extremely sensitive,6,000
very reproducible and quantitatively precise. 400
4,000
2,000 Zoom 200
0 0
Quantification for G1F glycopeptide EEQYN[+1606.6]STYR 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Replicate injection Replicate injection

×10 2 ×10 4
Blank 2 Amount of total mAb %RSD
Counts

2.48
1.9 digest
EEQYN(+1606.586706)STYR
on-column (ng) (n = 10) % Accuracy
2.46 1.8 y = 943.671754*x – 5.093398
1.7 0.0197
R = 0.99814103
2 18.5 97.5
×10 2 1.6
0.0394 18.3 104.2
0.0098 ng 1.5
Counts

2.48
1.4 0.0591 12.5 108.3
2.46 1.3
1.2 0.0985 9.3 102.4
×10 2 1.1 ×10 2
Response

0.0197 ng 1.0 0.197 13.0 95.6

Counts

0.9 0.394 3.0

2.5 5.9 84.8 2.6
0.8
0.7 0.985 5.7 102.1 2.2
Response

×10 2 0.6 1.8

0.0394 ng 0.5 1.97 1.6 105.5 1.4
Counts

0.4 1.0
2.6 0.3 19.7 2.9 99.7
0.6
0.2 Zoom 0.2
×10 2 0.1 -0.2
0.0591 ng 0 0 0.04 0.08 0.12 0.16 0.20 0.24 0.28
Counts

-0.1
2.6
-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Concentration (ng of mAb digest on-column)
0.90 0.95 1.00 1.05 1.10
Acquisition time (min)

20,000 932.7 & 204.1

For Research Use Only. Not to be used in diagnostics procedures. 1,400
18,000 932.7 & 2,227.9
16,000 932.7 & 2,065.8 1,200
14,000 932.7 & 1,392.6
Response

Response

1,000
12,000
932.7 & 366.1 800
10,000
8,000 600
6,000 400
4,000
2,000 Zoom 200
0 0
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Replicate injection Replicate injection

×10 4
2
1.9 EEQYN(+1606.586706)STYR
1.8 y = 943.671754*x – 5.093398
4 1.7 R2 = 0.99814103
1.6
1.5
1.4
1.3
1.2
e
 Food Testing – Pesticides

Evaluation of Optimized Inter-MRM Delay Times

The global nature of food commodities has prompted increased scrutiny concerning safety and origin. Recent contamination issues stemming
from use of illegal pesticides and adulterants have negatively affected consumer confidence. To meet this challenge, Triple-Quadrupole LC/MS is
used to simultaneously monitor hundreds of potential contaminants.
Here, an MRM method was developed targeting mycotoxins, illegal dyes and pesticides in spices. The exceptional sensitivity afforded by the
Agilent 6495B Triple Quadrupole LC/MS system enabled precise and accurate quantitation of these excipients, with reduced matrix effects and
improved method robustness.

×10 6

Tribenuron-methyl
2.5 396.0 & 155.1
Peak area at 0.5 ms
2.0 = 0.80
Metobromuron Peak area at 5.0 ms
Counts

259.0 & 179.0

1.5
Peak area at 0.5 ms
= 0.94
1.0 Peak area at 5.0 ms 5.0 ms
─
─ 2.0 ms
0.5 ─ 1.0 ms
─ 0.5 ms

0
8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8
Acquisition time (min)
Overlaid MRM chromatograms of metobromuron and tribenuron-methyl (100 ppb) at dwell times of 5.0, 2.0, 1.0, and 0.5 ms using optimized inter-MRM delay times.

5
 ANALYTICAL CONFIDENCE

Environmental Water Analysis

Accuracy and Precision for Analysis of PPCPs in Water

Worldwide, scientists are encountering trace amounts of Pharmaceuticals and Personal Care Products (PPCPs) in our drinking water. Typically,
compounds found are extremely diverse, and may contain; drugs of abuse, spices, petrochemicals, active ingredients form pharmaceuticals and
their metabolites. Each geographical region has a PPCP profile uniquely its own. These active ingredients, byproducts and metabolites are often
not completely removed during the sewage-treatment process. That means the drug-tainted wastewater can enter groundwater and surface
water, which are collectively the major sources of drinking water for a wide variety of species.
The high accuracy, precision and sensitivity of the Agilent 6495B Triple Quadrupole LC/MS system enabled direct injection of water samples
without prior sample pre-concentration. The results show screening and quantitative analysis of 32 PPCPs in surface water samples at levels that
extend to 0.5 ng/L.

×107
×105
A B
5.5 Diclofenac Venlafaxine
1.8
5.0 y = 546.946323*x – 34.491767 y = 36438.173667*x – 15563.567037
R2 = 0.99954397 1.6 R2 = 0.99762610
4.5
4.0 1.4
3.5 1.2 OH
Cl
Response

Response

3.0 1.0
2.5 0.8 N
NH
2.0 0.6
Cl OH
1.5
0.4
1.0 O
0.2 O
0.5
0 0
-0.5 -0.2
0 100 200 300 400 500 600 700 800 900 1,000 0 50 100 150 200 250 300 350 400 450 500 550
Concentration (ng/L) Concentration (ng/L)

Calibration plots for diclofenac (negative mode) and venlafaxine (positive mode) in water evaluated for quantitation accuracy and peak area RSD.

6
 Environmental Water Analysis

×103
2.4 A
2.2 Atrazine
10 replicate injections Quant.: 216.1 & 174.1
2.0
0.5 ng/L Qual.: 216.1 & 146.0
1.8 RSD = 6.5 % (Quant)
1.6 IDL = 0.10 ng/L ×102
7 0.1 ng/L
1.4 6
Counts

5
1.2 4
1.0 3
2
0.8 1
0.6 0
3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0
0.4
0.2
0
3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0
Acquisition time (min)
1.3 B
1.2 Diltiazem
10 replicate injections Quant.: 415.2 & 178.1
1.1
0.5 ng/L Qual.: 415.2 & 150.0
1.0 RSD = 8.1 % (Quant)
×102
0.9 IDL = 0.12 ng/L 6
0.8 5 0.1 ng/L
4
Counts

0.7
3
0.6
2
0.5 1
0.4 0
0.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0

0.2
0.1
0
2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00
Acquisition time (min)

Overlaid MRM chromatograms for two example PPCPs evaluated in this study at 0.5 ng/L. Reproducible
responses (RSD% <10 %) were observed for both quantifier and qualifier ions of atrazine (A) and diltiazem (B)
at sub-ng/L concentrations.

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 PERFORMANCE SPOTLIGHTS

Ion optics and ion detector

Enhanced Sensitivity and Precision Yields the Lowest Limits of Detection and Quantitation
Greater ion sampling and ion transmission efficiency yields better assay performance. Superior ion optics design, along with enhanced detector
electronics have drastically improved the sensitivity of the 6495B LC/TQ system. This means better detectability and improved precision at low
analyte levels.

x101
Area %RSD = 15.1 x101 Area %RSD = 12.4
6.6
6.7 n = 10 injections 6.4
n = 7 injections
6.6
6.5 %Accuracy = 116 6.2 %Accuracy = 85.1
6.4 6.0
6.3
6.2 5.8
6.1 5.6
6.0 5.4
5.9 5.2
5.8
5.7 5.0
5.6
LLOQ of Verapamil 4.8
LLOQ of Bentazon
3.62 3.66 3.7 3.74 3.78 3.82 3.86 3.9 3.94 3.98 4.02 4.06
40 attogram on-column 4.6 1 femtogram on-column
Counts vs. Acquisition Time (min) Blank 4.4 Blank
2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6
Counts vs. Acquisition Time (min)

8
Instrument Detection Limit (IDL)
Today’s “low-noise” mass spectrometry systems demand a statistical standard that measures the true sensitivity of the system; a specification
proportional to ion count. Agilent is leading the way with an innovative method to effectively determine system performance – Instrument
Detection Limit (IDL)

Estriol amount measured Replicates %RSD t(99%) Estriol IDL • IDL is a rigorous metric based on statistical
5 pg/mL (LLOQ) n = 7 injections 11.3 3.143 1.8 pg/mL analysis of precision (%RSD)
MDL = t x (%RSD/100) x Amount = 3.143 x (10.4/100) x 25 fg = 1.8 pg/mL • Measured at or close to the detection limit (LOD)
• Provides an accurate assessment of the system’s
x101
7 Inj1
x101
7 Inj3
x101
7 Inj5
x101
7 Inj7 true detection limit and LLOQ
44.17 41.16 50.07 52.23
6 6 6 6
• A uniform and practical means of evaluating
5 5 5 5
sensitivity performance
x101 x101 x101
7 Inj2 7 Inj4 7 Inj6 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4
Counts vs. Acquisition Time (min)
More Ions = Better Precision
53.76 45.90 54.05
6 6 6
5 5 5

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4
Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min)

MassHunter software
High Sensitivity MRM at the Fastest Analysis Times
The analysis of multiple compounds in targeted applications requires the ability to intelligently schedule multiple MRMs while maintaining high
sensitivity at short MRM dwell times. Dynamic MRM sets optimal dwell times to allow confident analysis of a large panel of compounds with
enough data points across the peak for precise and accurate quantitation.

x104
2.0 123 Pesticides, Carbosulfan,
1.9
1.8 1ng/mL 18 data points
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5
Counts vs. Acquisition Time (min)

9
COMPREHENSIVE SOLUTIONS

Where solutions come together

The best solutions don’t come together by accident. Decades of experience designing mass spec systems gives us a
clear advantage, but we refuse to rest on reputation. We continually engage in groundbreaking collaborations with
thought leaders in chemistry and the life sciences, and we never stop learning from longstanding partnerships with
analytical laboratories in every field.
Whatever you need to analyze, we have the know-how—and the connections—to direct you to the best approach
and help you implement it.

1290 Infinity II LC system

A mass spec this powerful requires the best performing Liquid Chromatograph. Agilent offers the
most comprehensive portfolio of analytical LC Systems optimized for unparalleled performance
when interfaced to Mass Spec. The 1290 Infinity II LC represents the next generation LC for
ultra-high performance liquid chromatography with superior reliability and is the perfect match for
Agilent’s LC/MS portfolio including the 6495B Triple Quadrupole LC/MS system.
Each 1290 Infinity II module is optimized to deliver the highest level of efficiency from sample
introduction to separation and detection as well as maximizing sample capacity, fastest injection
cycles and pump performance. The 1290 Infinity II LC coupled with Agilent’s LC columns and
supplies offers the most comprehensive solutions for LC/MS available today.

Efficient Sample Preparation and Columns

Agilent Bond Elut SPE and QuEChERS sample preparation provide rugged, reliable sample clean-up
to minimize matrix interferences and reduce sample related system maintenance, improving system
performance and analytical sensitivity. InfinityLab Poroshell 120 columns provide exceptional
efficiency, speed and resolution for reliable, reproducible results. Twelve chemistries, including
HPH-C18 for high pH applications, allow you to achieve the optimal separation for any sample.

10
High quality MS application solutions
Simplify your startup and quickly set up a method for your
specific application
Veterinary drug tMRM database
Curated database with over 650 compounds, with up to 10 MRM
transitions, fragmentor voltages, and collision energies for each
compound. Instantly build methods for targeted screening and
confident quantitation for hundreds of analytes in a single run.
MassHunter software
Agilent MassHunter Workstation software simplifies sample
management, MS method optimization, data processing, and data
reporting for quantitative analyses. An impressive suite of tools
facilitates sample analysis, particularly for routine quantitation.
These tools include:
• Optimizer software—delivers automated determination of MRM
transitions for quantitation and optimized collision energies for
each analyte

Pesticides tMRM database for triple
quadrupole LC/MS
Agilent's pesticide tMRM database for LC/TQ systems contains over
750 compounds, with up to 10 MRM transitions, fragmentor voltages,
and collision energies for each. Instantly build methods for targeted
screening and confident quantitation for hundreds of analytes in a
single run.
• Dynamic MRM (dMRM)—ensures the best possible quantitative
results for multi-analyte assays compatible with fast UHPLC
separation, by specifying cycle times and allowing the software to
determine the maximum dwell time for each MRM transition
• Triggered MRM (tMRM) data-dependent acquisition—allows fast
analyte quantitation together with compound confirmation at the
lowest assay levels

Agilent CrossLab
You can trust Agilent CrossLab service experts to deliver valuable insights and keep your instru-
ments running at top performance. Our industry-leading services—tailored to meet your needs—
include instrument transition, application consulting, repairs, preventive maintenance, compliance
verification, and education. Ask us how we can support your laboratory today.

11
Any analyte. Any method. One partner: Only Agilent covers all your mass spec needs
Whatever you need to analyze, we are here to help you implement the ideal method to achieve your goals. Agilent
has decades of experience designing and building chromatography and mass spectrometry systems. Our longstanding
partnerships and collaborations span every field of application. This is what enables Agilent to provide you with the
best solution for your analytical needs.

Learn more
www.agilent.com/chem/6495B

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www.agilent.com/chem/store

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in your country
www.agilent.com/chem/contactus

USA and Canada

1-800-227-9770
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This information is subject to change without notice.

© Agilent Technologies, Inc. 2017

Printed in the USA January 10, 2017
5991-4541EN