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C&EN PRESENTS:

Improving Liquid Chromatographic

Separations in Clinical Analysis by
Utilizing Recent Developments in
Column Technology

BROUGHT TO YOU BY:

 Improving Liquid Chromatographic Separations
in Clinical Analysis by Utilizing Recent
Developments in Column Technology
Abstract
Liquid chromatography (LC) is a powerful technique
for the separation, identification, and quantification of
target analytes in complex media such as biomatrices.
LC offers target assay flexibility, multiple detection
modes, and can be highly automated — all of these
are ideal features to be applied to the analysis of high
sample numbers or for research purposes in the modern
clinical laboratory. New stationary phase technology
has provided further benefits for the clinical analyst by
offering columns with multiple mechanisms of interaction
to maximize separation selectivity, and therefore, improve
analyte resolution and lab productivity. Finally, with the
introduction of ever-more-powerful and easier-to-use
mass spectrometry (MS) detectors at an affordable
price point, LC-MS has become increasingly influential
as a routine, investigative tool for clinical testing,
therapeutic/dose monitoring, and the screening of
disease states including quantitative and qualitative
biomarker profiling.
Introduction
Different forms of chromatography have been applied
for many years in the clinical laboratory; these include
high performance LC (HPLC), gas chromatography (GC),
and thin layer chromatography (TLC). With appropriate
sample work up, these separation techniques allow
the quantification of specific target analytes within
the complex biological matrices that are routinely
seen in clinical labs. LC has become the most widely
established of these techniques and is now routinely
used in various applications such as therapeutic drug
monitoring (TDM) and specific low-molecular-weight
analyte determinations that include catecholamines,
metanephrines, steroid hormones, vitamins, etc. [5].
Detection techniques include UV, fluorescence (FLD),
electrochemical (ECD), or MS. A variety of other wet
chemistry/biochemistry analytical techniques are still
routinely used in clinical labs and include the well-known
enzyme-linked immunosorbent assay (ELISA). ELISA
approaches are still common and especially useful for
large biomolecule detection such as proteins, peptides,
antibodies, etc. However, as LC hardware technology,
‘total solutions,’ and separation performance for these
analytes improve, the increased biomolecule knowledge
provided by the high-resolution LC-MS approach is
likely to drive further adoption of the technique over
the legacy ELISA methodology.
1 mac-mod.com
LC-MS in the Clinical Laboratory
Over the last 15–20 years, MS has become firmly
established as a powerful detection mode used in
clinical LC. In some specialist clinical laboratories,
LC-MS has been adopted as the instrument of choice
to replace well-established GC (-MS) and HPLC (-UV,
-ECD, -FID) methods [4]. The major advantages of LC-MS
include the ability to detect and quantify multiple target
analytes in a single run (multiplexing), low reagent
costs, and the flexibility for users to quickly develop
custom assays. Excellent specificity and sensitivity
can be achieved by operating the LC-MS system in
selective reaction monitoring (SRM) mode [1]. In SRM,
a product ion from the parent target analyte molecule is
isolated and fragmented to generate multiple product
ions, which can be individually monitored as distinct
ions specific for that target analyte. Monitoring these
“transitions” is highly selective toward the target analyte
and almost entirely eliminates interference from other
sample components (although ion suppression from
co-eluting matrix components such as phospholipids
and salts is an important issue that must be suitably
assessed during assay development). LC-MSn systems
can monitor multiple transitions sequentially using
the multiple reaction monitoring (MRM) mode, thus
permitting the simultaneous determination of multiple
analytes and internal standards within the same
analytical run. Figure 1 shows a 51-analyte drugs-of-
abuse screen using an ACE Excel C18, 1.7 μm UHPLC
column. The high performance of the ACE UHPLC
column provides excellent resolving power for this
wide range of analytes, while the triple quadrupole
MS detection provides excellent specificity and
sensitivity for the measurement of individual target
analytes. A similar method has also been developed
using the ACE Excel C18-PFP phase to identify and
quantify 250 drugs of abuse in urine samples [7].
 Figure 1

Conditions
35
Column: ACE Excel C18, 1.7 μm 100

Dimensions: 100 x 2.1 mm

Part Number: EXL-171-1002U
Mobile Phase: A: 5 mM ammonium acetate in H2O
B: 5 mM ammonium acetate in MeOH
Time (mins) %B 48
14
0.0 10 37, 38, 39
51

10.0 90
11.9 90

%
13.4 10
34
15.5 10 17 20 41 46,47
Flow Rate: 0.3 mL/min 5

Injection: 10 µL 40 42

Temperature:
45
40 °C 9 12 18 21 28 32 44
30
Detection: MS Quattro Premier XE triple quad 10 36
43
25 26,27
MRM, positive and negative ESI mode
Desolvation temperature: 450 °C 2 4 6
13 15 19 24
29
33

Ion source temperature: 150 °C 1

3
7
8 11
16 23 31 49,50
Collision gas pressure: 3.5 x 10-3 mbar 0
22
Time, min
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Figure 1: UHPLC-MS/MS screen of 51 drugs of abuse using an ACE Excel C18, 1.7 µm column. Reproduced with permission of Biotage GB
Ltd, UK. The full list of analytes is available in ACE Application Note #AN2190. View at: mac-mod.com/_applications-and-analytes-id-260.php

Some of the potential disadvantages of LC-MS include:

Conditions
the high degree of technical experience required to Column: ACE C18-AR, 3 μm
Dimensions: 50 x 2.1 mm
operate the instrumentation and to interpret data Part Number: ACE-119-0502
and results compared to other techniques, the potential Mobile Phase: A: 10 mM ammonium acetate in H2O
B: 10 mM ammonium acetate in MeOH
lower flexibility for open access [1,3], and the high initial
capital investment and ongoing service costs [3]. Other Time
%B
(mins)
techniques such as immunoassay may often be easier 0 75
1. Itraconazole
(m/z 705.3 à 392.3)
to integrate into the lab, require less user expertise to 2 98
3 98
run, and are cheaper when performed in low volume.
Although immunoassays show excellent automation Flow Rate: 0.7 mL/min
Temperature: 45 °C
capabilities, harmonization of methodologies, and ease Detection: AB Sciex 4000 3. Hydroxyitraconazole 4
(m/z 721.3 à 408.2) (
of use, they often show potential for interference ESI positive ion mode
1. Itraconazole 2. Itraconazole-d5
Sample: 1. Itraconazole
1.0 ng/mL human
(m/z 705.3 àwhole 2. Itraconazole-d5
392.3) blood (LLOQ) (m/z 710.4 à 397.4)
(cross-reactivity) and have high reagent costs. (m/z 705.3 à 392.3) (m/z 710.4 à 397.4)

Despite the above disadvantages, LC-MS is

becoming widely adopted and is beginning to 3. Hydroxyitraconazole 4. Hydroxyitraconazole-d5
(m/z 721.3 à 408.2) (m/z 726.4 à 413.3)
replace traditional analytical methods for many 3. Hydroxyitraconazole
(m/z 721.3 à 408.2)
4. Hydroxyitraconazole-d5
(m/z 726.4 à 413.3)
clinical analyses. Some of the earliest uses of LC- Figure 2
MS in the clinical setting were seen in newborn cps
cps
2
1 1.6e5
screening and therapeutic drug monitoring (TDM) 250

[2,4]. In the therapeutic drug monitoring setting, the 1.2e5

200
monitoring of immunosuppressants (e.g., tacrolimus,
cyclosporin, sirolimus, and everolimus) is one of the 150
8.0e4

most prominent applications [3,6]. LC-MS has also 100

been applied to the analysis of a range of drug 4.0e4

classes including antifungal drugs, antibiotics, and 50

antiepileptic drugs [6], etc. Figure 2 shows the 0

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
0.0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

analysis of the antifungal medication itraconazole and its

Time, min
Time, min

metabolite hydroxyitraconazole in whole blood using an 3 4

ACE Excel C18-AR, 3 µm column. This novel stationary 6.0e 4

phase combines the stability and retentivity of a C18 120

phase with phenyl functionality to provide unique

4.0e4
selectivity, suitable for the separation of structurally 80

similar analytes (see page 7 for further details). The high

specificity, sensitivity, and short run time achieved 40 2.0e4

makes the technique ideal for high-throughput

TDM testing, and it is no real surprise to see LC-MS 0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
0.0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

becoming a prominent technology in the pediatrics,

Time, min Time, min

endocrinology, and toxicology fields. Figure 2: Determination of itraconazole and hydroxyitraconazole in

whole blood using an ACE Excel C18-AR, 3 µm column. Reproduced
with permission of Agilux Laboratories, USA. View at: mac-mod.com/_
applications-and-analytes-id-118.php

2 mac-mod.com
 Recent Developments in Column Technology
and Its Application to Clinical Analyses
Along with the rapid expansion of the application of
LC-MS, new column technologies have been introduced
to complement the technique and to help meet the
demands of clinical analyses. One challenge in routine,
high-throughput testing is to reduce analysis time to
maximize sample throughput. In the clinical analysis
setting, this challenge is continually driven by high
year-on-year increases in sample number. Ultra-High
Performance LC (UHPLC) was commercialized in 2004
and uses smaller format columns (e.g., 3.0- and 2.1-mm
ID geometries) packed with sub-2-µm particles, typically
operated at pressures up to 1,000 bar or ~15,000 psi
[8,13]. These small particles have higher optimum flow
rates than those of larger diameter particles (3, 5,
and 10 µm) that are typically used in traditional LC.
Additionally, sub-2-µm particles show a flatter van
Deemter curve (measure of performance/efficiency
vs. flow rate/linear velocity), meaning that they still
deliver higher efficiency at supra-optimal flow rates.
In contrast, larger particles show a sharper drop-off in
performance when flow rates higher than the optimum
value are used [13]. The high efficiency and favorable
van Deemter characteristics of sub-2-µm particles allow
separations to be performed at elevated flow rates with
increased sensitivity to reduce the chromatographic run
time dramatically. The application of novel stationary
phase chemistries that maximize selectivity in UHPLC
(discussed on page 7) has further enhanced the
technique’s attractiveness.
Figure 3
the demands of UHPLC. The particles packed into
ACE UHPLC columns have been developed to be
mechanically stable enough to tolerate the very high
pressures of continued routine use. The packing
techniques used to manufacture ACE Excel UHPLC
columns have also evolved to ensure that high quality,
reproducible, and stable packed beds can be reliably
produced in UHPLC format columns. This allows
chromatographers to realize the high theoretical
efficiencies of sub-2-µm particles, while achieving
column lifetimes comparable to those obtained with
traditional HPLC. ACE Excel UHPLC columns are
packed using proprietary high stability column (HSCTM)
packing technology to ensure that columns are
robust and deliver excellent column lifetime under
UHPLC conditions.
Figure 3: Translation of a gradient method for the separation of six
peptides from HPLC to UHPLC.
Mobile phase A: 0.05% TFA in H2O
Mobile phase B: 0.05% TFA in MeCN
Temperature: 60 °C
Injection volume: 10 µL (150 x 4.6 mm column) and
1.4 µL (50 x 3.0 mm column)
Detection: UV, 220 nm
Sample: 1. Gly-Tyr, 2. Tyr-Tyr-Tyr, 3. Val-Tyr-Val, 4. Oxytocin,
5. Angiotensin II, 6. Leu-enkephalin
View at: mac-mod.com/_applications-and-analytes-id-320.php
Figure 3A

Both Figure 3A and 3B provide examples of how ACE mAU 1 ACE Excel C18, 5 μm, 150 x 4.6 mm
UHPLC technology can be applied to dramatically 700 RS = 3.1
Flow rate: 1.00 mL/min
Gradient:
increase sample throughput. Figure 3A shows an 600
2
Time (mins) %B
Figure 3 0 5
existing LC method for the separation of six peptides 500
3 17 40
6 23 90
using a traditional HPLC format reversed phase column 400
1 ACE Excel C18, 255 μm, 90 x 4.6 mm
150
mAU
(ACE Excel C18, 150 x 4.6 mm, 5 µm) and a 400 bar 300

700 RS = 3.1
Flow rate: 1.00 26
mL/min
46
5
5
200
4 5 Gradient:
HPLC system. The method was translated to an ACE 600
100 2
Time (mins) %B
0 5
Excel C18 UHPLC column (50 x 3.0 mm, 1.7 µm) and 500
0 3 17 40
run on a 1400-bar-maximum UHPLC system for an 400 0 5 10
6
15 20
23
25
90
25 min
90
analysis time of < 2 minutes and a total cycle time 300
mAU ACE Excel C18, 26 1.7 μm, 550 x 3.0 mm
600
200
4 5 Flow rate: 1.00 46
mL/min 5
of 6.5 minutes (Figure 3B). This method translation 100 Gradient:
500
Time (mins) %B
produced an 86% savings in run time and solvent Figure
0
3B RS = 3.0 0.00 5
400
consumption. Quantitative translation of this method 0 5 10 15 20
2.41
3.26
40
25 min
90
300
(i.e., the exact same separation and selectivity of the mAU ACE Excel C18, 3.541.7 μm,90 50 x 3.0 mm
600
Flow rate: 1.00 mL/min 5
3.69
rapid method compared to the original method) was
200
6.52 5
500
Gradient:
achieved using the free-to-download ACE LC Translator 100

RS = 3.0
Time (mins) %B
0.00 5
Tool [9]. These same principles can be applied to most
400
0 0 1 2
2.41
min
40
300 0 5 10 15 20 3.26 90
25 min
legacy and newly developed clinical separations to 3.54 90
3.69 5
significantly increase laboratory sample throughput. 200
6.52 5
100

The use of higher flow rates in UHPLC and the 0 0 1 2 min

concomitant higher operating back pressures are

0 5 10 15 20 25 min

usually above the ~400 bar pressure limit offered by

traditional HPLC instruments. Column manufacturing
technology has, therefore, also advanced to meet

3 mac-mod.com
 In addition to increasing the speed of analysis, the high
separation efficiency offered by UHPLC can be used to RT MRM ION
ANALYTE
achieve higher resolution by performing separations on (MINS) TRANSITIONS MODE
longer columns packed with sub-2-µm UHPLC particles.
This approach can be used to resolve closely eluting Aldosterone-d4 2.62 363.1 190.3 -
analytes and is ideally suited to the LC-MS analysis of Aldosterone 2.65 359.1 189.3 -
complex samples containing closely eluting peaks with Cortisone 3.03 359.1 189.3 +
similar chemical structures and m/z values. For example, 18-OH-Corticosterone 3.06 363.3 269.2 +
a 100 mm column packed with 1.7 µm particles will
Cortisol 3.51 363.4 121.3 +
offer twice the efficiency of a 50 mm, 1.7 µm column.
This approach is particularly useful for multi-analyte DHEAS 3.76 367.1 97.1 -
complex separations such as steroid panels. Steroid 21-Deoxycortisol 4.27 347.1 311.2 +
hormone analysis using immunoassays has long been Corticosterone 4.63 347.3 329.3 +
problematic due to interferences between steroids
11-Deoxycortisol 4.76 347.3 109.3 +
that are very structurally similar (for example, DHEA’s
Androstenedione 5.38 287.3 97.2 +
(dehydroepiandosterone) interference in testosterone
immunoassays) [6]. The use of LC-MS methods for such Estradiol 5.47 271.1 145.2 -
steroids provides a high specificity, high sensitivity Estrone 5.51 269.2 145.2 -
solution by separating these components and avoiding 11-Deoxycorticosterone 5.75 331.3 109.1 +
the potential for interference. The high separation
Testosterone 5.91 289.3 97.1 +
performance available with ACE UHPLC columns
offers a solution to this issue by providing sufficient 17-OH-Progesterone 6.11 331.3 97.1 +
resolving power to separate such structurally similar 17-OH-Pregnenolone 6.18 315.3 297.2 +
steroid analytes. Figure 4 shows the separation of 21 DHEA 6.23 289.3 253.2 +
key steroids, extracted from human serum, using an DHT-d3 6.80 294.4 258.3 +
ACE Excel C18, 1.7 μm, 100 x 2.1 mm UHPLC column.
DHT 6.82 291.3 255.3 +
By using a 100 mm UHPLC column, a high-resolution
separation of these structurally similar components is Progesterone 7.29 315.2 97.2 +
achieved, thus allowing their accurate determination Pregnenolone 7.89 299.3 159.3 +
and quantification by LC-MSn. For example, the isobaric
steroids, cortisol, and 18-hydroxycorticosterone, are Table 1: Steroid analytes, retention times, MRM transitions, and ion modes for
well separated, as are the isobaric testosterone and Figure 4
DHEA, along with the isobaric trio of 21-deoxycortisol,
corticosterone, and 11-deoxycortisol.
Figure 4

3.5x106 Conditions
Column: ACE Excel C18, 1.7 μm, 100 x 2.1 mm
Part Number: EXL-171-1002U
3.0x106 Mobile Phase: A: 0.2 mM ammonium fluoride in water
B: Methanol

2.5x106 Time (mins) %B

0 50
3 60
2.0x106 8 90
9 95
9.1 95
1.5x106
9.5 50

Flow Rate: 0.4 mL/min

1.0x106
Injection Volume: 10 µL
Temperature: 40 oC
5.0x105
Detection: Shimadzu 8060 Triple Quad MS
MRM, positive and negative ESI mode
Sample: Endogenous Steroids extracted from human
0 serum using supported liquid extraction or
polymer-based SPE prior to LC/MS-MS
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 mins

Figure 4: UHPLC-MSn analysis of 21 endogenous steroids from human plasma on an ACE Excel C18, 1.7 µm UHPLC column. Reproduced with
permission of Biotage GB Ltd. For sample extraction conditions, see Biotage Application Notes AN890 and AN891.

4 mac-mod.com
 The Use of Solid Core Technology for resistant to acid hydrolysis, and therefore, provides
Clinical Analysis exceptionally low column bleed, ideal for high sensitivity
For many laboratories, the capital investment in UHPLC LC-MS applications. Moreover, ACE UltraCore columns
instrumentation to help reduce sample analysis turn- can be readily applied using mid and high pH mobile
around times may not be an affordable option due to phases using volatile buffers commonly used with LC-
associated service costs plus staff training. In these MS. The ability to use mid and high pH mobile phase
cases, the use of solid-core particles (a relatively recent permits improved retention, peak shape, and loading
LC particle development), may offer a solution. The for basic analytes. ACE UltraCore columns are available
majority of modern LC phases are manufactured from with both SuperC18 and SuperPhenylHexyl bonding
fully porous, spherical silica particles. For example, ACE chemistries and can be used over an extended mobile
LC columns are packed with spherical particles based phase pH range of 1.5–11.0, thereby providing further
on ultra-high purity, fully porous silica, which provide flexibility to the clinical analyst.
excellent peak shape and efficiency and are ideal
for LC-MS applications. In recent years, solid-coreFigure 5
particle columns have been developed, such as the Partially porous shell
ACE UltraCore. These new products feature a solid,
non-porous core (Figure 5), surrounded by a porous
Non-porous core
outer shell. The particle morphology has been shown
to provide exceptional performance when compared to
fully porous particles at HPLC pressures (i.e., < 400 bar)
[15]. For example, ACE UltraCore 2.5 μm particles can
produce peak efficiencies similar to 1.7 µm UHPLC fully
porous particles and can also be operated at elevated
flow rates without sacrificing performance.

Importantly, the efficiency values observed for these

solid core particles do not result in a significant increase
in operating back pressures associated with sub-2-µm
totally porous UHPLC particles. As a result, columns
packed with 2.5 or 5 µm ACE UltraCore solid core
particles are compatible with standard 400 bar HPLC
systems and can be used to increase separation efficiency
and drive improvements in sample throughput. As a
general rule of thumb, 2.5 µm solid core particles give
similar efficiencies to sub-2-µm porous particles, while
5 µm solid core particles offer similar efficiencies to 3 µm
fully porous particles [15]. ACE UltraCore phases are
bonded using unique Encapsulated Bonding Technology
(EBTTM) to provide a stationary phase that is exceptionally
Figure 5: Morphology of ACE UltraCore solid core particles
In Figure 6, the use of the solid core technology with
MS detection provides a rapid separation of a range
of clinically useful catecholamines and metanephrines
in urine. Selective detection was achieved for the
individual analytes by MRM using a triple quadrupole
mass spectrometer. The exceptional separation
efficiency of the ACE UltraCore SuperPhenylHexyl
column provides excellent resolution for a rapid
1-minute gradient and a total gradient cycle time of just
4.5 minutes.

1 Conditions
3.75e6
Column: ACE UltraCore SuperPhenylHexyl, 2.5 μm, 100 x 2.1 mm
3.5e6 Part Number: CORE-25B-1002U
Mobile Phase: A: 2 mM ammonium formate + 0.05% formic acid in H2O
3.25e6
B: 2 mM ammonium formate + 0.05% formic acid in MeOH
3.0e6 6 Time (mins) %B
2.75e6 0.00 0
1.00 70
2.5e6 2
1.10 70
2.25e6
1.11 0
2.0e6 4 4.50 0
1.75e6
Flow Rate: 0.3 mL/min
1.5e6 Injection: 10 µL
1.25e6
5 Temperature: 30 °C
Detection: Shimadzu LCMS-8040
1.0e6 ESI in positive ion mode
Sample: Standard 100 ng/mL in urine (after SPE purification)
7.5e5
1. Norepinephrine 4. Dopamine
5.0e5 3 (m/z 170 à 107) (m/z 154 à 91)
2.5e5 2. Epinephrine 5. Metanephrine
(m/z 184 à 166) (m/z 198 à 180)
0.0
3. Normetanephrine 6. 3-Methoxytyramine
0.0 1.0 2.0 3.0 4.0 mins
(m/z 184 à 166) (m/z 181 à 91)

Figure 6: LC-MSn determination of catecholamines and metanephrines in spiked urine using an ACE UltraCore SuperPhenylHexyl 2.5 µm
column. Reproduced with permission of Shimadzu, France. View at: mac-mod.com/_applications-and-analytes-id-458.php

5 mac-mod.com
 The Application of Unique Selectivity
Stationary Phases for Challenging
Clinical Applications
The reliability, reproducibility, selectivity, stability, and A B 100 1

low stationary-phase-bleed characteristics of C18 phases ACE C18-PFP 50

have made them the most widely used column chemistry
0
for virtually all clinical LC applications. The mechanism of
separation for C18 columns is dominated by hydrophobic 100
No Column
interactions between the analyte and stationary phase.
While C18 phases can provide good separations in many 50

cases, there are many examples where other mechanisms

0
of interaction, provided by a different stationary phase,

Relative Abundance
Conditions
are required to deliver a separation that is not possible Column Dimensions: 50 x 3.0 mm
100

with a C18 phase. One of the many challenges with Mobile Phase: 5 to 95 %B in 5 min, hold at 95 %B for 5 min
A: 0.1 % formic acid in water 50
clinical analysis is the separation of analytes of interest B: 0.1 % formic acid in acetonitrile
Flow Rate: 0.43 mL/min
from interfering isomeric and isobaric species. In clinical Temperature: 60 °C 0
labs, the requirements of fast analysis and the use of Detection: Agilent 1100 MSD positive ESI fast scan
mode, full scan 50 – 1000 m/z 100
“standard” C18 stationary phases has led to limitations Sample: Blank runs

in the ability to separate closely related analytes from 50

each other. As the complexity of analyses performed on
clinical samples increases with parallel gains in instrument Figure 7A: Demonstration of the low LC-MS bleed 0

sensitivity, there is the potential to discover more characteristics of the ACE C18-PFP
100
unresolved natural isomers [10].
50
:
: : 0

B 100 1 100 6 Conditions 0 1

B B100 100 1 1 100 100
80 6 6 Conditions
Conditions
Column: ACE Excel C18-PFP, 2.0 μm,
50 80 80
50 50
60 Column:
Column: ACE 100
ACE
Excel
xExcel
C18-PFP,
2.1 mmC18-PFP,
2.0 μm,
2.0 μm,
60 60 100 x100
2.1 xmm
2.1 mm
40 Part Number: EXL-1010-1002U
0 40 40
0 0 20 Part Number:
Part Number:
Mobile Phase: EXL-1010-1002U
EXL-1010-1002U
A: 0.1% formic acid in H2O
20 20
0
Mobile
Mobile
Phase:
Phase: A: 0.1%
A: 0.1%
B: 0.1%
formicformic
formic
acid in H2O
acid
acid in H2O
in MeOH
100 2 0 0 B: 0.1%
B: 0.1%
formicformic
acid in
acid
MeOH
in MeOH
100 100 2 2 Time (mins) %B
50
100 7 TimeTime
(mins)(mins)
0.0
%B %B
0.5
50 50
100 100
80
7 7 0.0 0.0 0.5 0.5
80 80
0.5 0.5
60 0.5 0.5 0.5 0.5
0 9.0 90.0
0 0 60 60 9.0 9.0 90.0 90.0
40 13.0 90.0
Abundance

Abundance

40 40
100 3 13.0mL/min
13.0 90.0 90.0
Relative Abundance

Abundance

20 Flow Rate: 0.3

Relative Abundance

Abundance

B for 5 min
100 100 3 3 20 20
0 Flow Flow
Rate:Rate:
Sample: 0.3 mL/min
0.3 mL/min
Dried serum extract
Bmin
for 5 min 50 0 0 Sample:
Sample:
Detection: DriedPositive
Dried
serumserum
extract
mode extract
ESI
e 8 Detection:
Detection: Positive
Positive
modemodeESI ESI
Relative

50 50
Relative

e
100 8 8
Relative

Relative

100 100
0 80
st scan 0 0 80 80
60
nst scan
100 4 60 60
100 100 4 4 40
1. Carnitine 6. Hexanoylcarnitine
40 40
20 1. 162.16
(m/z Carnitine
1. Carnitine
→ 85.25-85.35)6. (m/z
Hexanoylcarnitine
6.260.20
Hexanoylcarnitine
→ 85.25-85.35)
50 20 20
50 50 0 (m/z 162.16
(m/z 2.
162.16
→ 85.25-85.35)
→ 85.25-85.35)
Acetylcarnitine (m/z 260.20
(m/z7.260.20
→ 85.25-85.35)
→ 85.25-85.35)
Octanoylcarnitine
0 0 2. Acetylcarnitine
2. Acetylcarnitine
(m/z 204.13 → 85.25-85.35)7.(m/zOctanoylcarnitine
7.288.20
Octanoylcarnitine
→ 85.25-85.35)
0
100 9 (m/z 204.13
(m/z
3. 204.13
→ 85.25-85.35)
→ 85.25-85.35)
Propionylcarnitine (m/z 288.20
(m/z
8.288.20
→ 85.25-85.35)
→ 85.25-85.35)
Myristoylcarnitine
0 0
100 100 9 9 3. (m/z
Propionylcarnitine
3. 218.13
Propionylcarnitine
→ 85.25-85.35)8. (m/z
Myristoylcarnitine
8.372.30
Myristoylcarnitine
→ 85.25-85.35)
100
80 5 (m/z 218.13
(m/z
100 100 5 80 80
60 5 4. 218.13
→ 85.25-85.35)
→ 85.25-85.35)
Butyrylcarnitine & (m/z 372.30
(m/z
9.372.30
→ 85.25-85.35)
→ 85.25-85.35)
Palmitoylcarnitine
60 60
4. Butyrylcarnitine
4.Isobutyrylcarnitine
Butyrylcarnitine
& & 9. (m/z
Palmitoylcarnitine
9.400.30
Palmitoylcarnitine
→ 85.25-85.35)
40 Isobutyrylcarnitine
50
40 40 (m/zIsobutyrylcarnitine (m/z 400.30
232.15 → 85.25-85.35) (m/z 400.30
→ 85.25-85.35)
→ 85.25-85.35)
50 50 20 (m/z 232.15
(m/z 232.15
→ 85.25-85.35)
→ 85.25-85.35)
5. Isovalerylcarnitine &
20 20 5. Isovalerylcarnitine
5. Isovalerylcarnitine
& &
0 0 2-Methylbutyrylcarnitine
0 00 1 2 3 4 5 6 7 08 0 6 7 8 9 10 11 12 13 2-Methylbutyrylcarnitine
2-Methylbutyrylcarnitine
(m/z 246.20 → 85.25-85.35)
0 1 0 2 1 3 2 4 3 Time
5 4 6(min)
5 76 87 86 76 87 98 109Time11
(min) 12
10 11 13
12 13(m/z 246.20
(m/z 246.20
→ 85.25-85.35)
→ 85.25-85.35)
Time (min)
Time (min) Time (min)
Time (min)

Figure 7B: The application of ACE C18-PFP application for acylcarnitine analysis in dry serum extract.

Reproduced with the permission of Department of Pathology, Immunology and Laboratory Medicine, University of Florida, USA.
View at: mac-mod.com/_applications-and-analytes-id-215.php

6 mac-mod.com
 The use of the latest in highly selective stationary phase
technology to assist with analyte resolution is, therefore,
very desirable.
Widely available and popular reversed-phase, non-C18
stationary phase chemistries include the following
phase types: alkyl aromatic phases (e.g., phenylpropyl,
phenylhexyl, and pentafluorophenyl propyl (PFP))
and polar-embedded phases (e.g., embedded amide
and carbamates). However, most, if not all, of these
shorter chain phases can have shortcomings such as
shorter column lifetimes, unacceptably high phase
bleed, or reproducibility issues that will be undesirable
for developing robust and reproducible methods. The
potential for stationary phase bleed, particularly
for stationary phases bonded with shorter, more
chemically labile ligands, is highly undesirable for
LC-MS analysis, because it can cause reduced and/or
variable signal sensitivity.
To overcome the undesirable characteristics of the
shorter chain phases, several novel ACE chemistries
were designed, which combine the ruggedness and
stability of traditional C18 bonded phases with the
alternative selectivity provided by other functional
groups. The application of this rational-phase-design
approach has resulted in the development of five
novel chemistries that include the ACE C18-AR, ACE
C18-PFP, ACE C18-Amide, ACE CN-ES, and ACE
SuperC18 phases. The combination of novel selectivity
with the longer chain, rugged, and stable C18 ligand
reduces stationary phase bleed and provides superior
LC-MS compatibility. For example, in Figures 7A and
7B, the low bleed characteristics of the ACE C18-
PFP stationary phase and its applicability for the
LC-MSn analysis of acylcarnitines in dried serum extract
are demonstrated.
These novel phases incorporate added functionality
within the C18 bonded phase ligand to provide
substantially different selectivity to traditional C18
BONDED PHASE
Hydrophobic
π-π Interaction
Binding
ACE C18 ****
ACE C18-AR ****
ACE C18-PFP ****
ACE SuperC18 ****
ACE C18-Amide ****
ACE CN-ES ***
Approximate value – determined by semi-quantitative mechanism weightings and/or by reference to other ACE phases using >100 characterizing analytes.
1
Table 2: Separation mechanisms provided by the ACE C18 phase and the five novel phases.
7 mac-mod.com
phases for a particular separation. For example,
the ACE C18-AR incorporates aromatic functionality
into a C18 ligand to afford enhanced interactions
with electron-deficient, aromatic analyte functional
groups. Conversely, the ACE C18-PFP incorporates
the pentafluorophenyl functional group into a C18 ligand,
which provides increased interactions with analytes
having electron-rich aromatic groups. The ACE C18-
Amide was designed with a longer spacer between the
silica and the embedded amide group, to provide
added stationary phase stability along with the unique
selectivity of a polar-embedded, amide phase. The ACE
CN-ES was designed not only to provide alternative
CN selectivity, but also to specifically address the
low stability of traditional short-spacer CN columns.
By employing an extended alkyl spacer between
the silica and the cyano group, the ACE CN-ES
stability is significantly improved over traditional
cyanopropyl phases. Finally, the ACE SuperC18 utilizes
novel encapsulated bonding technology (EBT) to
extend the usable pH range of this phase and allows
the development and use of methods over a broad pH
range (1.5–11.5).
The characteristics of the ACE novel chemistries are
summarized in Table 2. All ACE column chemistries have
been designed and characterized using data from the
Tanaka protocol [14] – a well-established and accepted
approach to understanding stationary phase retention
mechanisms. The data from these five tests can help
the analyst understand the weightings of different
mechanisms that contribute to analyte retention for
each column chemistry.
An example of the power of using different stationary
phase selectivity is illustrated with fat-soluble vitamin
analysis. Vitamin D2 and D3 are hydroxylated by the
liver to 25-hydroxy vitamin D2/D3, the most abundant
circulating form. This biologically inactive form is then
further hydroxylated by the kidneys to the biologically
active form, 1,25-dihydroxyvitamin D2/D3.
Separation Mechanism and Relative Strength1
Hydrogen Shape
Dipole-Dipole
Bonding Selectivity
- - * **
*** (donor) * ** ***
*** (acceptor) **** *** ****
- - - **
- ** **** **/***
* *** ** *
 Conditions
Column: ACE Excel C18-PFP, 2.0 μm, 100 x 2.1 mm

Serum Vitamin D testing is one of the most important Part Number: EXL-1010-1002U
Mobile Phase: A: 2 mM ammonium acetate, 0.1% formic acid in H2O
applications of LC-MS in the clinical lab and targets the B: 0.1% formic acid in MeOH
Time (mins) %B
most abundant 25-hydroxy forms to determine vitamin 0.0 75

D status. A major challenge in vitamin D measurements 3.0

4.0
100
100
Flow Rate: 0.4 mL/min
is the ability to separate the isobaric 3-epi-25 hydroxy Injection: 15 µL
Temperature: 40 °C
forms of vitamin D3 from the 25-hydroxy variant. Detection: Quattro Premier XE triple quad MS
MRM positive ESI mode
Although the physiological relevance of the 3-epi form Desolvation temperature: 450 °C
Ion source temperature: 150 °C

of vitamin D3 is presently unclear [12], it is believed to 100

be a biologically inactive or a suppressing form [4], 25-OH Vitamin D2

%
(m/z 395.5 à 269.5)
and can be present in samples from infant patients. 0
2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00

Separation of the 3-epi isomer could therefore be 100

25-OH Vitamin D2
useful for the determination of 25-hydroxyvitamin D3

%
(m/z 395.5 à 119.2)
0
in infants [11 and references therein]. As the 3-epi and 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00

25-hydroxy species are isomeric and isobaric, these two 100

d6-25-OH Vitamin D3 (IS)

%
analytes typically co-elute on standard C18 phases (not 0
(m/z 389.6 à 263.5)

enough selectivity) and cannot be distinguished using 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00

100
LC-MS n (identical mass and fragmentation patterns). 25-OH Vitamin D3

%
(m/z 383.5 à 257.5)
Using the power of novel stationary phase selectivity, 0
2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
however, it is possible to separate the two forms using 100

the ACE C18-PFP phase, as shown in Figure 8A. The 25-OH Vitamin D3

%
(m/z 383.5 à 107.2)
added PFP functionality of the ACE C18-PFP permits 0
2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90
Time
3.00

near baseline resolution of the two analytes, thus

allowing their accurate determination and quantification Figure 8B: Application of the ACE Excel C18-PFP to the LC-MSn
for this challenging application. Figure 8B shows the determination of 25-hydroxy vitamin D2 and D3 in serum (Reproduced
applicability of a 100 x 2.1 mm, 2 µm, ACE C18-PFP for the with permission of Biotage GB Ltd. For extraction conditions, see
determination of 25-hydroxy vitamin D2 and D3 in serum. Biotage Application Note AN842).
As clinical applications of LC and LC-MS continue to evolve, http://mac-mod.com/_applications-and-analytes-id-276.php
such novel LC stationary phase chemistries will likely prove
to be a powerful tool available for the clinical analyst Conclusions
searching for separation solutions to a wide range of Over the last 15–20 years, LC and LC-MS have proved
structurally similar analytes. to be powerful techniques for a wide range of clinically
relevant analytes in a variety of complex sample
matrices. Applications range from rapid assays of single
Conditions
Column: ACE Excel C18-PFP, 3.0 μm, 50 x 4.6 mm analytes for dose monitoring to complex screening
Part Number: EXL-1110-0546U
Mobile Phase: MeOH:H2O 83:17 + 0.1% formic acid
experiments targeting tens or even hundreds of
Flow Rate: 1 mL/min 2. 3-epi-25-hydroxy vitamin D3 individual components. As the use of LC and LC-MS in
Injection: 10 µL
Temperature: 22 °C clinical applications grows, improvements in instrument
Detection: UV, 265 nm
Instrument: ChromasterUltra Rs and column technology enable the analyst to perform
Figure 8
Sample: 0.25 µg/mL of 25-hydroxy vitamin D3 and
3-epi-25-hydroxy vitamin D3
more complex analyses, while increasing sample
throughput and reducing sample turnaround times.
A 1. 25-hydroxy vitamin D3 The introduction of UHPLC and sub-2-µm UHPLC particle
technology enables both existing and new methods to be
Conditions
accelerated and provides
Column: ACE Excel 3opportunities
C18-PFP to achieve higher
Dimensions: 50 x 4.6 mm
resolution
Part Number: separations for challenging applications
EXL-1110-0546U
withPhase:
Mobile
Flow Rate:
increased sensitivity.
MeOH:H2O 83:17
1 mL/min
In+ 0.1%
recent years, the new
formic acid

developments in ACE reversed-phase chemistry have

Injection: 10 µL
Temperature: 22 °C
1 provided a range ofUV,novel
Detection: 265 nm selectivity phases that enable
2 the separation
Instrument:
Sample:
of closely related
ChromasterUltra Rs analytes and isobaric
0.25 µg/mL of 25-hydroxy vitamin D3 and
species, which couldn’t have been
3-epi-25-hydroxy achieved previously
vitamin D3

using standard C18 phases. The introduction of solid-

Figure 8A: Separation of 25-hydroxy vitamin D3 and its 3-epi form
using the novel ACE Excel C18-PFP stationary phase.
View at: mac-mod.com/_applications-and-analytes-id-335.php
core technology along with broad-pH-range phases
permits high resolution separations for both UHPLC and
HPLC applications in the clinical laboratory. As LC-MS
continues to be used for new applications in the clinical
setting, these novel ACE stationary phases will provide
the analyst with a powerful set of tools for the ever-more-
1. 25-hydroxy vitamin D3 2. 3-epi-25-hydroxy vitamin D3
challenging clinical analyses encountered.

8 mac-mod.com
 References
1. “Applications of HPLC in clinical diagnostics” (published on clinlabint.com, 2006, https://www.clinlabint.com/fileadmin/
pdf/pdf_general/applications-of-hplc-in-clinical-diagnostics.pdf)

2. M. J. P. Wright, S. Hepburn, LCGC Europe 34 (2016) 37–41

3. J.E. Adaway, B.G. Keevil, L. J. Owen, Annals of Clinical Biochemistry 52 (2015) 18–38

4. ACE Application note AN4140 “Drugs of Abuse Screen (250 Analytes) in Urine by LC-MS/MS” in “ACE Complete
Applications Guide”, page 69, http://www.ace-hplc.com/announcements/ace-application-guides.aspx or http://mac-mod.
com/pdf/product-bulletins/ACE%20Complete%20Applications%20Guide.pdf

5. G. Brandhorst, M. Oellerich, G. Maine, P. Taylor, G. Veen, P. Wallenmacq Clinical Chem 58 (2012) 821–825

6. S. K. G. Grebe, R. J. Singh Clin Biochem Rev 32 (2011) 5–31

7. J. E. Adawaya, B. G. Keevila Journal of Chromatography B 883-884 (2012) 33-49

8. M. W. Dong, LCGC North America 35 (2017) 374–381

9. ACE Knowledge Note #AKN0011 “Practical Ultra High Performance Liquid Chromatography (UHPLC)”.

10. http://mac-mod.com/resources-tools.php

11. ACE Knowledge Note #AKN0019 “ACE UltraCore and Solid Core Technology”.

12. V. M. Carvalho Journal of Chromatography B 883-884 (2012) 50–58

13. K. Kimata, K. Iwaguchi, S. Onishi, K. Jinno, R. Eksteen, K. Hosoya, M. Arki, N. Tanaka, J. Chromatogr. Sci. 27 (1989) 721

14. S. E. Berger, M. I. Van Rompay, C. M. Gordon, E. Goodman, M. Eliasziw, M. F. Holick, J. M. Sacheck Appl. Physiol. Nutr.
Metab. 43 (2018) 259–265

15. D. Thibeault, N. Caron, R. Djiana, R. Kremer, D. Blank Journal of Chromatography B 883-884 (2012) 120–127

9 mac-mod.com