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RECENT
Volume 34, Number s4 April 2016
www.chromatographyonline.com
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DEVELOPMENTS IN LC COLUMN
TECHNOLOGY
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Recent Developments in LC Column Technology: Impact on a World of Disciplines . . . . . . . . . . . . . . . 8David S. BellA brief introduction of the articles presented in this supplement.
The Impact of Superficially Porous Particles and New Stationary-Phase Chemistries on the LC–MS Determination of Mycotoxins in Food and Feed . . . . . . . . . . . . . . . . . . . 10Andreas BreidbachThis fit-for-purpose LC–MS-based method provides fast analysis of four mycotoxins using
standard HPLC equipment with a pentafluorophenyl SPP column.
The Synthetic Cannabinoid Chemical Arms Race and Its Effect on Pain Medication Monitoring . . . . . 15Sheng Feng, Brandi Bridgewater, Gregory L. McIntire, and Jeffrey R. EndersAn investigation of C18 and phenyl-hexyl column chemistries for definitive identification
of 13 synthetic cannabinoid metabolites in patient samples.
HPLC Column Technology in a Bioanalytical Contract Research Organization . . . . . . . . . . . . . . . . . . 24Ryan Collins and Shane NeedhamWhen presented with a new analyte, a bioanalytical CRO must quickly develop a robust method with good chromatographic
resolution, repeatable results, and a quick run time. Recent developments in LC column technology make that possible.
Characterizing SEC Columns for the Investigation of Higher-Order Monoclonal Antibody Aggregates . . . 28Ronald E. Majors and Linda L. LloydWhen selecting the optimum phase for SEC separations, several key column parameters must be considered carefully.
Positive Impacts of HPLC Innovations on Clinical Diagnostic Analysis . . . . . . . . . . . . . . . . . . . . . . . 37Michael J.P. Wright and Sophie HepburnAs clinical diagnostic assays move to LC–MS-MS, the emphasis has turned to emerging stationary phases that
use alternative mechanisms of retention to separate the analyte–interference critical pairs.
Latest Advances in Environmental Chiral Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Denise WallworthRecent advances in chiral stationary phases have enabled higher efficiency and faster separations in studies of the differing
enantiomeric activity of pesticides, their environmental transformation, and the degradation of pollutants in general.
Cover Imagemore Co, Ltd./Stocktrek Images/Andrew Brookes/Liz Pedersen/EyeEm/GIPhotoStock/Andy Sacks/Arne Pastoor/Getty Images
Articles
Apr i l 2016
Volume 34 Number s4
6 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016
Recent Developments in
LC Column
Technology
Recent Developments in
LC Column
Technology
www.chromatographyonline.com
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8 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
FROM the GUEST EDITOR
Recent Developments in LC Column Technology: Impact on a World of Disciplines
There have been many advances in liquid chromatography (LC) during the past
decade. Much attention has been paid to the development of new and improved
particle designs to achieve higher efficiency and there have been many new
developments in the surface treatments of these particles that impact retention and
selectivity. Novel particle designs such as sub-2-μm and superficially porous media
have vastly improved the speed and efficiency of separation tasks. Newly developed
chemical modifications and their implementation using these modern particle archi-
tectures have greatly expanded their utility. The underlying theme for this special
supplement edition was to bring together articles that discuss how these innovations
have impacted analysis across a wide variety of disciplines.
Andreas Breidbach from the European Commission, Joint Research Center at the
Institute for Reference Materials and Measurements provides insight on how mod-
ern technologies have impacted the liquid chromatography–mass spectrometry (LC–
MS) analysis of mycotoxins in food and feed. The work demonstrates the increased
efficiency garnered from the use of superficially porous particles as well as added
selectivity through modern surface chemistry modifications. Sheng Feng and col-
leagues from Ameritox provide examples of similar achievements for the analysis of an
ever-growing number of synthetic cannabinoids for toxicology and forensic analyses.
Again, superficially porous particles combined with alternative surface chemistries
has enabled rapid, selective, and sensitive LC–MS-MS identification of 13 synthetic
cannabinoids in patient urine samples. Collins and Needham from Alturas Analytics
discuss the impact of recent column technology advancements and emerging devel-
opments in microflow LC technologies with respect to improving productivity in
the bioanalytical contract research realm. The authors note that these technologies
facilitate the development of robust and reliable methods, which may lead to lowering
the cost of complex biotherapeutics. Continuing with the theme of bioanalysis, Lloyd
and Majors discuss the importance of particle architecture and surface treatments
with respect to current needs in size-exclusion chromatography (SEC). The growing
attention of the pharmaceutical market on biotherapeutics has necessitated the imple-
mentation of many modes of chromatography to fully characterize these complex
systems. The authors point out the importance of particle pore size (and distribu-
tion), pore volume, and surface chemistry treatments as it pertains to modern SEC
requirements. From the world of clinical diagnostics and testing, Wright and Hep-
burn provide examples of how modern particle technologies, surface modifications,
and multiple-channel high performance liquid chromatography (HPLC) instruments
have enabled faster analyses for various disease states and patient types. This is a
crucial step toward providing high-quality health care. Lastly, Wallworth highlights
some of the recent advances in chiral stationary phases (CSP) and how they impact
important environmental concerns. Chirality plays a significant role in the study of
pollutants, agrochemical usage, and pharmaceutical waste on our environment. The
author anticipates that recent applications of CSPs on modern particle designs will
positively impact research in this arena.
In applications ranging from food to pharma and biotherapeutics to biomes,
advances in liquid chromatography are playing a critical role. Modern particle designs
and surface chemistry treatments are continually being adopted in a variety of dis-
ciplines. As exemplified by the articles within this supplement, developments in our
craft are improving the quality of life around the world. Enjoy!
David S. Bell
LCGC “Column Watch” editor
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10 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Andreas Breidbach
The Impact of Superficially Porous Particles and New Stationary-Phase Chemistries on the LC–MS Determination of Mycotoxins in Food and Feed
Superficially porous particles with their favorable chromatographic
properties were a great advance for liquid chromatography (LC).
Analytical LC columns packed with those particles allow for much
faster separations even with standard LC equipment rated at a
maximum pressure of 400 bar. This speed is exemplified by a LC–mass
spectrometry (MS) method of analysis for four mycotoxins, spanning log
P values from -0.7 to 3.6, with an analysis time of just over 8 min and
excellent performance. Another issue is the separation of closely related
mycotoxins, like 3- and 15-acetyldeoxynivalenol. With the common C18
chemistries, they are coeluted and identification and quantification can
only be achieved through differing MS-MS signals. Now, with the newer
pentafluorophenyl chemistries these two mycotoxins can be separated
by LC and MS quantification of them has become much more precise.
In 2006, high performance liquid
chromatography (HPLC) columns
packed with superficially porous
particles (SPP) (also known as porous-
shell, core–shell, and solid-core parti-
cles) were introduced to the market. In
performance rivaling sub-2-μm technol-
ogy, SPP packed columns have enabled
highly efficient separations to be car-
ried out with standard HPLC systems
because of the much lower back pres-
sure they generate (1). This favorable
characteristic has also been exploited
for the determination of mycotoxins in
food and feed.
Mycotoxins are secondary metabo-
lites of certain fungi whose occurrence
in food and feed is difficult to avoid.
Therefore, many countries have regu-
lated this occurrence of mycotoxins
(2,3). A wealth of methods of analysis
to enforce these regulations exist (4)
and among them liquid chromatog-
raphy–mass spectrometry (LC–MS)-
based detection is gaining momentum.
LC–MS is primarily gaining momen-
tum for two reasons: sample preparation
requirements can be relaxed because of
the high specificity and sensitivity of
MS detection, and multiple mycotoxins
can be determined in one go. Both of
these reasons are of particular interest
to official control laboratories since they
will lead to higher throughput compared
to traditional one analyte per prepara-
tion and run approaches with extensive
cleanup. This higher throughput has
been shown for traditional HPLC equip-
ment with an analytical column packed
with fully porous particles by Biselli
and colleagues (5). Using a 150 mm ×
2.1 mm column with 3-μm particles at
1-mL/min f low, 18 mycotoxins could
be detected during a 15-min analytical
run. With those settings, deoxyniva-
lenol (DON) eluted at 3.80 min and
zearalenone (ZON) at 7.38 min. To stay
within the operational envelope of their
electrospray ionization (ESI) source the
column eff luent was split 1:5. Using a
sub-2-μm fully porous particle packed
column of 100 mm × 2.1 mm dimen-
sions, Varga and colleagues (6) were able
to show a multimycotoxin separation
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 11www.chromatographyonline.com
in which DON eluted at 1.45 min and
ZON at 6.44 min with a total run time
of 11.5 min. To perform this separation,
an ultrahigh-pressure liquid chromatog-
raphy (UHPLC) system capable of deliv-
ering flows at pressures as high as 1200
bar was used.
With the desire to determine mul-
tiple mycotoxins in one run, the
necessity arose to be able to separate
closely related mycotoxins. One such
example would be DON and its two
acetylated relatives, 3- and 15-acetyl-
deoxynivalenol (AcDON). Although
DON can be separated from the two
AcDONs on a C18 column, the two
AcDONs are coeluted. Because of dif-
ferent fragmentation behavior it is still
possible to obtain individual quanti-
tative data using MS-MS detection,
but with lesser confidence than with a
full chromatographic separation (5). A
more recent stationary phase chemis-
try capable of separating such isomers
is the so-called pentaf luorophenyl
(PFP, F5) modified silica. The pentaf-
luorphenyl system is electron deficient
and can interact with the analyte in
multiple ways: π-π, dipole-dipole, and
charge-transfer interactions. Because
of these multiple interactions, struc-
tural isomers can often be separated.
This article presents a fit-for-purpose
LC–MS-based method of analysis for
the four mycotoxins DON, HT-2 toxin,
T-2 toxin, and ZON utilizing standard
HPLC equipment with an SPP column.
Performance characteristics in unpro-
cessed cereals, as determined in-house
and verified through a collaborative
trial, were in line with traditional single
analyte methods with a short analysis
time of under 9 min. The article also
shows how the F5 stationary phase
chemistry enables the separation of
the closely related mycotoxins 3- and
15-acetyldeoxynivalenol.
Experimental
Chemicals and Materials
All chemicals were purchased from either
Sigma-Aldrich or VWR and were of at
least analytical grade. For the mobile
phase LC–MS Chromasolv-grade (Fluka,
Sigma-Aldrich) water and methanol
were used. Deionized water was gener-
ated by a MilliQ system (Millipore). All
tested materials came from the material
pool of the European Union Reference
Laboratory (EURL) for mycotoxins at
the Institute for Reference Materials
and Measurements (IRMM) of the Joint
Research Centres (JRC) of the European
Commission (EC).
The mycotoxins DON, HT-2, T-2,
ZON, 3-AcDON, and 15-AcDON,
and the isotopologues 13C15-DON, 13C22-HT2,
13C24-T2, and 13C18-ZON
were purchased from Biopure (Romer
Labs) as either solids or ready-to-use
solutions. From these, a stock solution
of 3.2-μg/mL DON, 0.5-μg/mL HT-2
toxin, 0.3-μg/mL T-2 toxin, and 0.3-μg/
mL ZON in neat acetonitrile was pre-
pared and stored. This stock solution
was freshly diluted for every calibration
task. An internal standard solution with
the same concentrations of the respec-
tive 13C-isotopologues in neat acetoni-
trile was also prepared and used undi-
luted. These solutions were stable for at
least three months in the dark at 2–8 °C.
Equipment
Measurements were performed on
an LC–MS system consisting of two
LC‐20AD pumps (Shimadzu, high-
pressure binary gradient), an Accela
autosampler (Thermo Scientific), and
a TSQ Quantum Ultra triple-quadru-
pole mass spectrometer with an Ion-
Max HESI2 interface (both Thermo
Scientif ic). For analytical columns
either an Ascentis Express C18 (75
mm × 2.1 mm, 2.7-μm particle size,
Supelco, Sigma-Aldrich), a Kinetex
C18, or a Kinetex PFP (both 100 mm
× 2.1 mm, 2.6-μm particle size, Phe-
nomenex) were used. The gradient con-
ditions with the Ascentis Express C18
column were as follows: 0 min, 8% B;
2 min, 57% B; 6 min, 61% B; 6.1 min,
95% B; 7.6 min, 95% B; 7.7 min, 8%
B; 8.7 min, 8% B with mobile-phase A
consisting of 999:1 (v/v) water–formic
acid and mobile-phase B consisting of
999:1 (v/v) methanol–formic acid at
a f low rate of 0.3 mL/min. The col-
umn was maintained at 40 °C during
analysis. This nonintuitive gradient
was designed with optimal resolution
and shortest analysis time for just the
four mycotoxins in mind. For the two
Kinetex columns more-generic gradi-
ent conditions were used: 0 min, 8%
B; 8 min, 95% B; 8.1 min, 8% B; 10
min, 8% B at a column temperature
of 50 °C. The mobile phases and f low
Table I: MS source and analyzer settings. (The segment run times relate to
the Ascentis Express C18 column; for the Kinetex columns they were adjust-
ed to the respective retention times of the analytes.)
Item Segment 1 Segment 2 Segment 4
Run time (min) 0–2.6 2.6–4.9 4.9–8.7
Analyte DON + AcDON +
13C15-DON
HT2 + 13C22-HT2,
T2 + 13C24-T2
ZON + 13C18-ZON
Adduct Protonated Sodium Deprotonated
Transitions (collision energy [eV])
297A231 (16),297A249 (13),339A213 (20),339A261 (20),312A263 (9),312A276 (9)
447A285 (22),447A345 (20),469A300 (19),469A362 (18),489A245 (30),489A327 (25),513A260 (26),513A344 (23)
317A131 (25),317A175 (22),335A185 (26),335A290 (21)
Tube lens (V) 80 110 80
Polarity Pos Pos Neg
Spray voltage (V) 2800 2800 2000
Vaporizer temperature (°C) 350
Sheath gas pressure (arbitrary units)
30
Auxiliary gas pressure (arbitrary units)
10
Transfer capillary temperature (°C)
320
-
12 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
rate were as stated above. The MS sys-
tem settings can be found in Table I.
The data acquisition was segmented to
limit the number of acquired transi-
tions and enable longer dwell times
per segment.
Sample Preparation
In an appropriately sized tube, 2 g of
unprocessed cereal (comminuted to
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 13www.chromatographyonline.com
Repeatability was determined with
naturally contaminated materials at
three different contamination levels.
Near the low end of the calibration
range, the relative repeatability stan-
dard deviations (RSDr) were between
11% and 18% for the four analytes.
Toward higher contamination lev-
els, which were smaller than exist-
ing (DON and ZON) or anticipated
(HT-2 and T-2) legislative limits in
the European Union (EU), these val-
ues improved to ≤9%. Two of those
materials, the lowest and the high-
est contaminated, were also tested on
eight different days by three different
operators to determine intermediate
precision, or within laboratory repro-
ducibility. For the low contaminated
material relative intermediate preci-
sions (RSDi) were between 13% and
25% for the four analytes. For the
high contaminated material they were
between 11% and 17%. All of these
f indings were comparable with the
results of the collaborative trial (9).
As already mentioned, these per-
formance characteristics are quite
satisfactory considering the analysis
time is only 8.7 min. This is signifi-
cantly shorter than the analysis times
reported by Biselli (5) or Varga (6).
Figure 1 shows a typical chromato-
gram of the four analytes, which span
log P values from -0.7 (DON) to 3.6
(ZON). The narrow peaks with a
baseline width of ≤0.2 min attest to
the high efficiency of the SPP parti-
cles packed in a 75-mm column. Even
though a mobile phase with metha-
nol–water was used, the back pressure
during analysis never exceeded 230
bar, which is well below the maximum
pressure of standard HPLC equipment.
Compared to this, analysis time of the
same material in a different labora-
tory during the collaborative trial on
a 150-mm column packed with fully
porous particles takes more than twice
as long (20 min) with larger baseline
peak widths between 0.4 and 0.9 min
(Figure 2). Thus, the SPP column
provides superior resolution at shorter
analysis times.
The benefits of short analysis times
are obvious: higher throughput and
lower solvent consumption. Benefits
of the better resolution might not be
so obvious. Matrix effects in LC–MS
measurements inf luence ionization
eff iciency caused by, amongst other
things, coeluted compounds. Because
of the high specificity of MS, particu-
larly MS-MS, coeluted compounds,
more likely than not, will be unde-
tected. Better resolution will limit
possible coelution and, therefore, min-
imize inf luences on ionization eff i-
ciencies and maximize the ability of
unbiased determination. Furthermore,
in our case, the better resolution comes
from narrower and, hence, taller peaks,
which has a positive effect on limit of
detection and quantification.
To show how a stationary phase chem-
istry change helps in obtaining better
and more confident results, a maize
sample highly contaminated with DON,
AcDONs, and ZON was analyzed with
two columns with identical SPPs but
different chemistries, namely the Kine-
tex C18 and PFP columns. Figure 3
shows the two total ion chromatograms
(TICs). Even though the two AcDONs
were not separated with the C18 chem-
istry, they were with the PFP chemistry.
Retention for all analytes was slightly
higher on the PFP column. Because of
the different fragmentation behavior
of the two AcDONs in MS-MS the
contamination level of the individual
AcDONs can be estimated even from
peaks 2 and 3 in Figure 3b. But because
of significant overlap of the product ions,
this estimation comes with an increased
uncertainty. It goes without saying that
a separation as shown in Figure 3a is
absolutely preferable.
Rela
tive a
bu
nd
an
ce
Time (min)
RT: 11.92
RT: 10.29
RT: 14.09
18.009.108.385.643.052.69 16.2514.61
RT: 5.97
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
00 2 4 6 8 10 12 14 16 18
Figure 2: Total ion current of the same QC sample as in Figure 1. Run times: DON, 5.97 min; HT-2, 10.29 min; T-2, 11.92 min; ZON, 14.09 min. Column: 150 mm × 2 mm, 4-μm dp Synergi Hydro-RP (Phenomenex).
-
14 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Conclusions
Through the use of an SPP packed col-
umn, a short method of analysis for four
mycotoxins in cereals was developed that
is fit for the purpose of official food and
feed control. The total run time was 8.7
min for the mycotoxins DON, HT-2,
T-2, and ZON spanning log P values
from -0.7 to 3.6. Despite the short run
time, excellent resolution was obtained
with very satisfactory performance char-
acteristics. Method recoveries were indis-
tinguishable from 1 for HT-2, T-2, and
ZON. For DON a recovery of 0.83 was
determined and results for DON should
be corrected for this recovery level. Val-
ues of RSDr were 18% or smaller for
low contamination levels and improved
to 9% or smaller toward higher levels,
which were still below existing or antici-
pated EU legislative limits. Because of
the intelligent use of stable isotopologues,
matrix effects were negligible at a mini-
mal cost per sample.
Changing the stationary-phase chem-
istry from C18 to pentaf luorophenyl
enabled the separation of the structural
isomers 3- and 15-acetyldeoxynivalenol
as well as DON and ZON in a natu-
rally contaminated maize sample. This
stands to show that SPP-packed col-
umns and new stationary-phase chemis-
tries have advanced mycotoxin analysis
in food and feed.
Acknowledgments
The author would like to thank Katrien
Bouten, Kati Kröger, and Karsten
Mischke for their excellent technical
support during method validation and
the collaborative study. The highly con-
taminated maize was a courtesy of the
Austrian National Reference Laboratory
for mycotoxins (AGES, Linz, Austria).
Disclaimer
Any trade names, trademarks, prod-
uct names, and suppliers named above
are only named for the convenience
of the reader of this publication and
their mentioning does not constitute an
endorsement by IRMM, JRC, or EC of
the products named. Equivalent prod-
ucts may lead to the same results.
References
(1) J.J. Kirkland, S.A. Schuster, W.L. John-
son, and B.E. Boyes, J. Pharm. Anal. 3(5),
303–312 (2013).
(2) Food Quality and Standards Service
(ESNS). Worldwide regulations for myco-
toxins in food and feed in 2003. 2004;
Avai lable from: http://www.fao.org/
docrep/007/y5499e/y5499e00.htm.
(3) European Commission, Commission Reg-
ulation (EC) No 1881/2006 of 19 Decem-
ber 2006 setting maximum levels for cer-
tain contaminants in foodstuffs (Text with
EEA relevance). Official Journal of the
European Union, 2006. L 364: p. 5–24.
(4) F. Berthiller et al., World Mycotoxin J. 8(1),
5–35 (2015).
(5) S. Biselli, L. Hartig, H. Wegner, and
C. Hummert, LCGC Europe Special Edi-
tion: Recent Applications in LC-MS 17(11a),
25–31 (2004).
(6) E. Varga et al., Anal. Bioanal. Chem.
402(9), 2675–2686 (2012).
(7) B.K. Matuszewski, J. Chromatogr. B
830(2), 293–300 (2006).
(8) A. Breidbach, Validation of an Analyti-
cal Method for the Simultaneous Deter-
mination of Deoxynivalenol, Zearalenone,
T-2 and HT-2 Toxins in Unprocessed
Cereals - Validation Report. 2011; Avail-
able from: http://skp.jrc.cec.eu.int/skp/
download?documentId=51161.
(9) A. Breidbach, K. Bouten, K. Kröger, J.
Stroka, and F. Ulberth, LC-MS Based
Method of Analysis for the Simultaneous
Determination of Four Mycotoxins in Cere-
als and Feed: Results of a Collaborative
Study (Publications Office of the European
Union, 2013). Available at: http://publica-
tions.jrc.ec.europa.eu/repository/bitstream/
JRC80176/la-na-25853-en-n.pdf.
Andreas Breidbach is with the European
Commission, Joint Research Centre, at
the Institute for Reference Materials
and Measurements in Geel, Belgium.
Direct correspondence to:
Rela
tive a
bu
nd
an
ce
Time (min)
11
2
2,3
3
4
(a) (b)
4
2.477.19 2.31
6.56
3.81
4.18
4.29
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Rela
tive a
bu
nd
an
ce
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
00 1 2 3 4 5 6 7 8 9 10
Time (min)
0 1 2 3 4 5 6 7 8 9
Figure 3: Total ion current of a maize sample highly contaminated with DON, AcDONs, and ZON; sample extract was diluted eight times; separation with (a) Kinetex PFP and (b) Kinetex C18 columns; Peaks: 1 = DON, 2 = 15-AcDON, 3 = 3-AcDON, 4 = ZON.
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 15www.chromatographyonline.com
Sheng Feng, Brandi
Bridgewater, Gregory L.
McIntire, and Jeffrey R. Enders
The Synthetic Cannabinoid Chemical Arms Race and Its Effect on Pain Medication Monitoring
In recent years, synthetic cannabinoids (“K2” or “spice”) have
experienced a boom in popularity. The negative health effects of these
drugs coupled with their increasing popularity led to placement onto
Schedule I by the Drug Enforcement Administration (DEA). In response,
the chemists behind these illicit compounds frequently invent new
compounds to circumvent the law. Thus, new classes and new examples
within classes of “spice” continue to become available for illicit use. In
this paper, we examine the use of two column chemistries (C18 and
phenyl-hexyl) in an effort to definitively identify synthetic cannabinoid
compounds in patient samples. Distinct synthetic cannabinoid
compounds interact differently with specific stationary phases and the
hope is that this extra dimension of data will help to rule out similar
interferent compounds that would otherwise cause false-positive results.
Synthetic cannabinoids, com-
monly known as “K2,” “spice,” or
“synthetic marijuana,” are often
sprayed onto or mixed with dried plant
materials and sold in convenience stores,
gas stations, smoke shops, and on the
internet. This ready availability causes
confusion about their safety and legality
(1). In recent years, synthetic cannabi-
noids have become increasingly popular
among adolescents and young adults as
one of several frequently abused sub-
stances. These synthetic drugs mimic
delta-9-tetrahydrocannabinol (THC),
but can be much more potent, which
results in psychoactive doses less than
1 mg (2). In fact, synthetic cannabi-
noids, which have a similar psychoactive
effect as cannabis, have strong addictive
properties often coupled with unknown
physiological impacts on users. A recent
study indicates that the use of synthetic
cannabinoids can be a cause of death (3).
Because of the high abuse potential
and lack of medical knowledge or usage,
these synthetic cannabinoids have been
added to the Schedule I list by the United
States Drug Enforcement Administra-
tion (DEA), as “necessary to avoid immi-
nent hazard to the public safety” (4). In
response, the chemists instigating this
illegal proliferation have synthesized
many new K2 analogs by slightly altering
chemical structures (5). Therefore, com-
pared with the relatively stagnant pool of
other compounds, such as opiates, that
most pain medication monitoring labo-
ratories deal with, the number of agents
on the list of synthetic cannabinoids has
been and continues to be increasing (6).
Testing for synthetic cannabinoids has
become a routine demand among pain
treatment clinics.
There are various types of synthetic
cannabinoids with different modifica-
tions on the core structure. The first
THC analogs, including HU-210 (7)
and CP-47, 497 (8), were synthesized in
the 1980s. Their inventions allowed the
discovery of G protein-coupled recep-
tors, CB1 and CB2 (9). Later on, a struc-
turally different analog, WIN55, 212-
2, was reported. Surprisingly, WIN55,
212-2 has higher affinity toward CB1
-
16 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
and CB2 than THC does (10). Subse-
quently, John W. Huffman developed
a series of “JWH compounds” by sim-
ply replacing the aminoalkyl group in
WIN55, 212-2 with simple alkyl chains
(11). JWH-018 has become the proto-
typical JWH compound. Synthetic can-
nabinoids have also been developed by
generating f luoro-derivatives of JWH
compounds. For example, AM-2201
and MAM-2201 are f luoro-derivatives
of JWH 018 and JWH 122, respec-
tively (12). By replacing the ketone in
the 3-indole position of JWH-018 with
an ester linkage, PB-22 and BB-22
compounds have been synthesized (13).
Furthermore, another class of synthetic
cannabinoids contains the tetrameth-
ylcyclopropyl ketone indoles, such as
UR-144 and its f luoro-derivative, XLR-
11 (14). Both UR-144 and XLR-11 have
cyclopropyl rings, and are therefore
likely to exhibit similar retention times
in liquid chromatography (LC).
The increasing number of sophisti-
cated reversed-phase LC separations has
led to the need for optimized stationary
phases to offer improved selectivity and
efficiency (15). In the present work, we
investigate C18 and phenyl-hexyl col-
umn chemistries for definitively identify-
ing 13 synthetic cannabinoid metabolites
in standards and patient samples.
Materials and Methods
Chemicals
Reference standards of AKB48
5-hydroxypentyl metabolite, AKB48
pentanoic acid metabolite, AM2201
4-hydroxypentyl metabolite, BB-22
3-carboxyindole metabolite, JWH-018
pentanoic acid metabolite, JWH-073
butanoic acid metabolite, JWH-122
5-hydroxypentyl metabolite, MAM-
2201 4-hydroxypentyl metabolite,
PB-22 3-carboxyindole metabo-
lite, PB-22 pentanoic acid metabolite,
UR-144 5-hydroxypentyl metabolite,
UR-144 pentanoic acid metabolite, and
XLR11 4-hydroxypentyl metabolite
were purchased from Cayman Chemi-
cal Company. Reference standards of
11-nor-9-Carboxy-Δ9-THC (THCA),
THCA glucuronide, and THCA-D9
were purchased from Cerilliant Cor-
poration. Solvents including methanol
(optima grade), acetonitrile (optima
grade), and formic acid (88%) were
purchased from VWR. Dimethylsulf-
oxide (DMSO) (HPLC grade), ethyl
acetate (optima grade), and ammonium
hydroxide (A.C.S. Plus) were purchased
from Fisher Scientific. Recombinant
β-glucuronidase enzyme was purchased
from IMCS. Drug-free normal human
urine (NHU) was purchased from
UTAK Laboratories, Inc. Deionized
(DI) water was obtained in-house from a
Thermo Scientific Barnstead Nanopure
water purification system.
HU-210
JWH-018 AM-2201 JWH-122 MAM-2201
PB-22 BB-22 UR-144 XLR-11
OH OH
OHOHH
O
O
O
O OO
OO
O OO
O
O
O
H
N
N
N
N NN
N
N
N
N N F
F
F
NH3C
H3C H3C
H3CH3C
H3CH3CH3C
CH3CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3 CH3
CH3
CH3CH3
CP-47, 497 WIN55, 212-2
XLR11 N-(4-hydroxypentyl) metabolite
UR-144 N-pentanoic acid metabolite
UR-144 N-(5-hydroxypentyl) metabolite
%B solvent
1 2 1 2
100
03 4 5
1 2 1 2 3 4 5
Time (min)Time (min)
C18
Rela
tive in
ten
sity
100
0
%B
%B
Phenylhexyl
Figure 1: Chemical structures of recent synthetic cannabinoids.
Figure 2: Total ion chromatography of 100 ng/mL calibrator in C18 and phenyl-hexyl columns with 2.5-min or 5-min methods. Red, blue, and green peaks represent XLR11 N-(4-hydroxypentyl), UR-144 N-pentanoic acid, and UR-144 N-(5-hydroxypentyl), re-spectively. Blue dashed lines indicate solvent gradients.
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 17www.chromatographyonline.com
Sample Preparation
Reference standards not already in solution were dissolved in
DMSO. Solutions of reference standards were aliquoted, dried,
and reconstituted with NHU to make a low calibrator concen-
tration at 1 ng/mL for all analytes except BB-22 3-carboxyin-
dole metabolite and THCA with low calibrator levels at 5 ng/
mL and 10 ng/mL, respectively. A high calibrator concentration
of 100 ng/mL in NHU was used for all analytes. An 18.5-ng/
mL THCA glucuronide hydrolysis–negative control (HNEG)
and a 20-ng/mL positive control (20CON) were similarly pre-
pared in NHU. This protocol uses THCA glucuronide as a
hydrolysis control. Accordingly, every curve and patient batch
has a hydrolysis control that contains 18.5 ng/mL of THCA
glucuronide. For this control to be considered passing, it must
return the expected THCA (parent) concentration within 30%.
Into 13 mm × 10 mm borosilicate glass tubes, 800 μL of
calibrators, controls, and samples were each aliquoted and com-
bined with 200 μL of THCA-D9 (2.5 μg/mL)/recombinant
β-glucuronidase (1000 enzyme units/mL) solution in 25:25:50
methanol–DI water–pH 7.5 phosphate buffer. All samples were
vortexed, transferred to SPEware CEREX PSAX 3 mL/35 mg
extraction columns in sample racks by SPEware, and heated
in a VWR Symphony oven for 15 min at 60 °C. Samples were
cooled for 5 min and placed on an automated liquid dispens-
ing-II (ALD-II) system for extraction. A light positive pressure
was applied to push the samples onto the solid-phase extraction
(SPE) packing. The ALD-II system then washed columns with
85:14:1 DI water–acetonitrile–ammonium hydroxide, washed
with 30:70 DI water–methanol, and finally eluted samples into
1800-μL amber autosampler vials using 98:2 ethyl acetate–for-
mic acid. Samples were dried under nitrogen for ~35 min at
25 °C in a SPEware Cerex sample concentrator, then each
reconstituted with 400 μL of 50:50 DI water–methanol. Sam-
ples were capped, vortexed for 20 s, and spun for 5 min at 4000
rpm on a Sorvall ST 40 centrifuge.
Patient Sample Collection
Patient urine specimens were collected at clinics and shipped to
Ameritox Ltd. These de-identified patient samples were treated
similarly to standards, that is, they were diluted, extracted, and
subjected to liquid chromatography–tandem mass spectrom-
etry (LC–MS-MS). Patient samples were selected for this study
Columnchemistry
C18
Co
un
tsC
ou
nts
Co
un
tsC
ou
nts
Pati
en
t 01
Pati
en
t 02
C18
Phenyl-hexyl
Phenyl-hexyl
JWH-018 N-pentanoic acidmetabolite qual372.2 → 126.9
1.8E4
1E4
0
1.6E4
0.8E4
0
1.2E4
0.6
0
9E3
4E3
0
1.8E4
1E4
0
0.4
1 1.2 1.4 1.6 1.8 1 1.2 1.4 1.6 1.8
1 1.2 1.4 1.6 1.8 1 1.2 1.4 1.6 1.8
0.6 0.8 1 1.2 1.4 0.6 0.8 1 1.2 1.4
1.2E4
0.6
0
1.2E4
0.6
0
Time (min) Time (min)
0.4 0.6 0.8 1 1.2 1.4 0.6 0.8 1 1.2 1.4Time (min) Time (min)
JWH-018 N-pentanoic acidmetabolite quant
372.2 → 155.1
IR fail5.4 ng/mL
IR pass14.5 ng/mL
IR fail5.4 ng/mL
IR pass14.5 ng/mL
Figure 3: Comparison of suspected JWH-018 pentanoic acid patient samples. The gray areas are integrated peaks. The dashed lines indicate the expected retention time based on the calibrators.
Columnchemistry
C18
Co
un
ts
Pati
en
t 02
Phenyl-hexyl
Time (min) Time (min)
MAM2201 N-(4-hydroxypentyl)metabolite quant
390.1 → 169.0
MAM2201 N-(4-hydroxypentyl)metabolite qual390.1 → 141.0
1E3
5E2
0
Co
un
ts
58
50
42
80
65
50
IR fail1.2 ng/mL
IR fail0 ng/mL
3.5E2
2.0E2
0.5E2
1.2
0.6 0.8 1 1.2 1.4 1.6 0.8 1 1.2 1.4 1.6
1.4 1.6 1.8 2 1.2 1.4 1.6 1.8 2
Figure 4: Comparison of suspected MAM-2201 metabolite pa-tient samples. The gray areas are integrated peaks. The dashed lines indicate the expected retention time based on the cali-brators.
( Keeping quality control under control. )
Amino acid analysis in accordance toEuropean Pharmacopeia 8.0
www.pickeringlabs.com
CATALYST FOR SUCCESS
PINNACLE PCX
-
18 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Table I: Mass spectrometry conditions for all methods in this study. The retention times coordinate with the 2.5 min
C18 and phenyl-hexyl method.
Compound NamePrecursor
IonProduct Ion
Fragmentation
(V)
Collision
Energy (V)
Cell Accelerator
(V)
C18 RT
(min)
Phenyl-hexyl
RT (min)
AKB-48 5-hydroxy-pentyl
382.11
107.00 380 52 2 1.99 1.24
92.90 380 60 2 1.99 1.24
135.10 380 10 5 1.99 1.24
AF4–MALS–dRI 396.1193.00 380 60 3 1.94 1.22
135.10 380 10 5 1.94 1.22
AM-2201 4-hy-droxypentyl
376.11
143.80 380 40 3 1.3 0.88
127.10 380 56 2 1.3 0.88
155.10 380 25 3 1.3 0.88
BB-22 3-carboxy-indole
258.01
118.00 380 24 5 1.8 0.97
54.90 380 36 2 1.8 0.97
175.90 380 10 7 1.8 0.97
JWH-018 N-penta-noic acid
372.21
126.90 380 60 2 1.36 0.94
55.00 380 56 2 1.36 0.94
155.10 380 25 3 1.36 0.94
JWH-073 butanoic acid
358.21
127.20 380 60 2 1.26 0.84
43.30 380 48 2 1.26 0.84
155.10 380 45 3 1.26 0.84
JWH-122 5-hydroxy-pentyl
372.11
115.10 380 72 4 1.65 1.11
169.10 380 21 4 1.65 1.11
141.00 380 55 4 1.65 1.11
THCA 345.20
327.20 380 18 2 2.1 1.31
299.20 380 18 6 2.1 1.31
193.20 380 18 2 2.1 1.31
MAM-2201 N-(4-hydroxypentyl)
390.11141.00 380 48 2 1.53 1.04
169.00 380 10 7 1.53 1.04
PB-22 3-carboxy-indole
232.01
118.00 380 16 2 1.53 0.75
43.10 380 24 2 1.53 0.75
132.00 380 10 7 1.53 0.75
PB-22 pentanoic acid
389.31
144.00 380 36 3 1.14 0.73
54.90 380 56 4 1.14 0.73
244.00 380 10 3 1.14 0.73
UR-144 5-hydroxy-pentyl
328.1155.00 380 44 2 1.74 0.93
125.00 380 10 3 1.74 0.93
UR-144 N-pentano-ic acid
342.11
125.00 380 20 3 1.68 0.92
54.90 380 48 4 1.68 0.92
244.00 380 10 4 1.68 0.92
XLR-11 4-hydroxy-pentyl
346.11143.90 380 44 3 1.49 0.79
248.00 380 20 2 1.49 0.79
THCA-d9 (internal standard)
354.10 336.10 380 13 5 2.09 1.29
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 19www.chromatographyonline.com
that were deemed positive by the current
method’s criteria, but were then deemed
negative upon closer manual inspection.
Instrumentation
All analyses were conducted by LC–
MS-MS on an Agilent 6490 triple-
quadrupole system run in electrospray
ionization (ESI) positive mode using
an Agilent 1290 chromatographic sys-
tem (1290 Inifinity binary pump, 1290
TCC, 1290 autosampler, and 1290 ther-
mostat) with a 100 mm × 2.1 mm, 2.7-
μm dp Agilent Poroshell 120 EC-C18 or
50 mm × 2.1 mm Phenomenex Kinetex
2.6 μm Phenyl-Hexyl column. Source
conditions were optimized with a
250 °C gas temperature, gas f low at
19 L/min, nebulizer set to 45 psi, sheath
gas heater at 300 °C, sheath gas f low
at 11 L/min, capillary voltage at 3.5
kV, and charging voltage at 2 kV. The
run time for this method is 2.21 min
with a cycle time of approximately
2.5 min. A longer chromatographic
method (roughly 5 min) was also used
in this study to help resolve question-
able interferences. All of these assays
monitor two or three transitions for
each of the following 14 analytes:
AKB48 5-hydroxypentyl metabolite,
AKB48 pentanoic acid metabolite,
AM2201 4-hydroxypentyl metabolite,
BB-22 3-carboxyindole metabolite,
JWH 018 pentanoic acid metabolite,
JWH 073 butanoic acid metabolite,
JWH 122 5-hydroxypentyl metabolite,
MAM2201 4-hydroxypentyl metabo-
lite, PB-22 3-carboxyindole metabo-
lite, PB-22 pentanoic acid metabolite,
UR-144 5-hydroxypentyl metabolite,
UR-144 pentanoic acid metabolite,
XLR11 4-hydroxypentyl, and THCA;
and one transition for one internal stan-
dard, THCA-D9. THCA is analyzed
by the mass spectrometer, but it is not
actively monitored in patient samples.
MS method parameters are shown in
Table I. The chromatographic start-
ing conditions are 40% mobile-phase
A (0.1% formic acid in 90:10 water–
methanol) and 60% mobile-phase B
(0.1% formic acid in methanol) with a
Table II: Gradient properties of the 2.5-min method
StepFlow Rate
(mL/min)
Time
(min)
%A (0.1% Formic Acid in
90:10 Water–Methanol)
%B (0.1% Formic
Acid in Methanol)
0 0.5 Initial 40 60
1 0.5 0.80 30 70
2 0.5 1.60 5 95
3 0.5 2.20 5 95
4 0.5 2.21 40 60
5 0.5 2.50 40 60
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20 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
0.5-mL/min f low throughout (Tables
II and III). The 2.5-min phenyl-hexyl
method was validated according to a
previously published procedure (16).
Results and Discussion
Various methods including colorimet-
ric detections (17), immunochemical
assays (18), nuclear magnetic resonance
(NMR) (19), gas chromatography–mass
spectrometry (GC–MS) (20), and LC–
MS-MS (21), have been developed for the
analysis of synthetic cannabinoids. With
those methods, many synthetic cannabi-
noids have been successfully analyzed in
different samples such as plant materi-
als, human hair, saliva, serum, and urine.
Several analytical reviews have summa-
rized the identification and quantifica-
tion techniques for synthetic cannabi-
noids that are currently popular (22,23).
Among those methods, LC–MS-MS
has clear advantages of ease and speed
of sample preparation and the capabil-
ity of automation. However, most of the
current methods only focus on a few
synthetic cannabinoids, or need a very
long chromatographic gradient to affect
resolution of spice compounds of inter-
est (usually longer than 10 min, see Table
IV). To improve the analysis of synthetic
cannabinoids, we developed new LC–
MS-MS methods with two different col-
umn chemistries (C18 and phenyl-hexyl),
which take either 2.5 min or 5 min for
each sample to achieve optimal resolu-
tion. These methods were applied to the
analysis of 13 synthetic cannabinoids.
We have analyzed a 100-ng/mL
synthetic cannabinoid calibrator that
includes all the K2 and spice com-
pounds of interest to this work with
both the 2.5-min or 5-min methods
in two different columns. Most of
the compounds were eluted in similar
order in the different columns, though
the elution time changed. Overall, the
compounds in the phenyl-hexyl column
are eluted earlier compared with ones
in the C18 column under both the 2.5-
min and 5-min methods, which may be
solely due to the shorter length of the
column or a combination of length and
selectivity. In addition, the three com-
pounds that share the tetramethylcyclo-
propyl ketone indole structural moiety
(that is, XLR11 N-[4-hydroxypentyl],
UR-144 N-pentanoic acid, and UR-144
N-[5-hydroxypentyl]) exhibit changed
elution order in the two different col-
umns. In both the 2.5-min and 5-min
methods, those three compounds were
eluted much earlier in order with the
phenyl-hexyl column compared to the
C18 column. This change in elution
order is not because of the change in the
column length. However, it might be
Table III: Gradient properties of the 5-min method
StepFlow Rate
(mL/min)Time (min)
%A (0.1% Formic Acid in
90:10 Water–Methanol)
%B (0.1% Formic
Acid in Methanol)
0 0.5 Initial 65 35
1 0.5 0.90 40 60
2 0.5 1.70 35 65
3 0.5 2.50 32 68
4 0.5 4.00 5 95
5 0.5 4.30 5 95
6 0.5 4.31 65 35
7 0.5 5.00 65 35
Table IV: LC–MS-MS conditions for synthetic cannabinoids in urine samples in
selected studies
Targets Purification ColumnTime of
Gradient
LOD
(ng/mL)Reference
Metabolites of JWH-018 and JWH-073
Dilution (hydrolysis)
Zorbax Eclipse XDB-C18 (150 mm × 4.6 mm, 5 μm)
10 min
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 21www.chromatographyonline.com
due to their tetramethylcyclopropyl structure having a higher
affinity toward the C18 column than for the phenyl-hexyl
column. Although this observation may seem trivial, it helps
illustrate the breadth of chemical components inherent in a
synthetic cannabinoid method. This challenge of chemical
breadth can be used as an advantage, however, if one con-
siders that synthetic cannabinoids with different chemical
structures will have different elution behaviors in two dis-
tinct column chemistries. In most cases, newly invented spice
compounds only slightly change the side chains of the banned
chemicals. It is possible that evaluating potential patient posi-
tives for this class of compounds using two different column
chemistries might help better separate compounds with simi-
lar chemical structures, thereby improving the detection of
novel compounds from existing agents.
These new methods for analyzing synthetic cannabinoids
were applied to suspected patient positive samples identified
from a production method. When the urine sample of patient
01, positive for JWH-018 pentanoic acid metabolite, was ana-
lyzed using both C18 and phenyl-hexyl columns, both quanti-
fier (quant) and qualifier (qual) peaks for JWH-018 pentanoic
acid metabolite came out earlier than expected based on cali-
brators (Figure 3). However, the ion ratio failed in the analysis
on the C18 column because of a missing qual peak, whereas the
ion ratio passed in the analysis with the phenyl-hexyl column.
Regardless of column chemistry, a human reviewer would likely
review this sample as negative or “unable to confirm” since
retention times do not perfectly line up. However, with the
phenyl-hexyl column data the peaks that passed the ion ratio
criteria were not all that far off with regards to retention time.
On a production floor it is not unreasonable for peaks to drift
0.3 min (18 s) over a given day or week, especially if this instru-
ment is used to run two different methods that may or may not
use different columns and solvents.
Meanwhile, in the test of patient 02, also potentially pos-
itive for JWH-018 pentanoic acid, all peaks showed up at
the expected retention times. The ion ratios passed on the
phenyl-hexyl column, but failed on the C18 column, which
is consistent with the result of patient 01. The data suggests
the phenyl-hexyl column significantly improved the detec-
tion of JWH-018 pentanoic acid metabolite in our methods
compared to the C18 column. The fact that this patient sam-
ple fails ion ratio (IR) on the C18 column and passes on the
phenyl-hexyl possibly indicates that an interferent coeluted
with one or both of the C18 peaks, thereby throwing off the
ion ratio. Cannabinoids (synthetic or otherwise), due to their
chemical makeup, are generally fat soluble and by extension
they also tend to be chromatographically coeluted with any
lipid content that may be in a sample. It is possible that this
interferent, which is throwing off the ion ratio in the C18 sam-
ple, is a lipid component that was able to survive the hydrolysis
and extraction protocol to be coeluted on the C18 column, but
on the phenyl-hexyl column it is sufficiently separated. It is
also possible that the compound from the patient sample is
isobaric with JWH-018 pentanoic acid and possesses the same
multiple reaction monitoring (MRM) transitions as JWH-018,
but at different ratios than the true calibrator compound. This
is possible if a small change in side chain configuration is envi-
sioned (for example, straight chain versus branched chain). The
technical and ethical issues associated with making a positive
call on such samples are not trivial.
Next, for a suspected MAM-2201 N-(4-hydroxypentyl)
metabolite, we found that patient sample 02 showed an
interfering peak, with slightly incorrect retention time, on
the C18 column. The chemistry of this interferent seems to
be drastically different compared to the MAM-2201 N-(4-
hydroxypentyl) metabolite, since it was not observed in the
Columnchemistry
C18
Pati
en
t 03
Phenyl-hexyl
Time (min)
UR-144 N-pentanoic acidmetabolite quant
342.1 → 125.0
UR-144 N-pentanoic acidmetabolite qual342.1 → 244.0
Co
un
tsC
ou
nts
7E3
3E3
0
5.0E3
2.5E3
0
IR fail5.2 ng/mL
IR fail4.9 ng/mL
4E4
2E4
0
3.0E4
1.5E4
0
1.2 1.4 1.6 1.8 2 1.2 1.4 1.6 1.8 2
0.6 0.8 1 1.2 1.4
Time (min)
0.60.4 0.8 1 1.2 1.4
Figure 5: Comparison of suspected UR-144 N-pentanoic acid patient samples. The gray areas are integrated peaks. The dashed lines indicate the expected retention time based on the calibrators. In this particular patient sample (when run on the C18 column), the actual qualifier peak was visible and chro-matographically separated; however, the integration software (under reasonable integration conditions) incorrectly selected the interferent for integration.
BensonpolymericTM
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22 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
window for the phenyl-hexyl column.
These types of interferences are ram-
pant among positive and questionably
positive synthetic cannabinoid patient
samples.
Patient 03 had a very strong well sepa-
rated quant peak for UR-144 N-penta-
noic acid, but the qual peak showed an
interferent just a few seconds away from
the targeted retention time. This inter-
ferent made detection of the qual peak
of interest very difficult. The qual peak
is still visible in the C18 separation;
however, the software (under reasonable
integration conditions) incorrectly inte-
grated the interferent. With the phenyl-
hexyl column chemistry, the qualifier
peak has coalesced into the interfer-
ent peak entirely and is not able to be
resolved, even with manual integration
intervention. The fact that this interfer-
ent moved proportionally with reference
to the expected UR-144 N-pentanoic
acid retention time indicates that this
interferent might share some chemical
functionality as discussed above.
Conclusions
A rapid, selective, and sensitive LC–
MS-MS method identifying 13 syn-
thetic cannabinoids in patient urine
samples has been described. Two dif-
ferent column chemistries (that is, C18
and phenyl-hexyl) have been applied
using this method. Three compounds,
including XLR-11 N-(4-hydroxylpen-
tyl), UR-144 N-pentanoic acid, and
UR-144 N-(5-hydroxylpentyl) metabo-
lites, demonstrate the different order
of elution on a phenyl-hexyl column
compared to the C18 column, while
most of the compounds maintain their
elution order. The fact that newly
invented synthetic cannabinoids often
only slightly change the side chains of
the banned drugs makes the detection
of those compounds more difficult. At
our laboratory, synthetic cannabinoids
are requested in roughly 20% of our
total samples and therefore should not
be written off as a fringe interest in
the pain medication monitoring arena
in spite of the very low positivity rate.
Using a second LC–MS-MS method to
confirm patient positives (as illustrated
here) is potentially useful for large scale
laboratories on a daily basis because of
the low positivity rates observed.
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Sheng Feng, Brandi Bridgewater,
Gregory L. McIntire, and Jeffrey R.
Enders are with Ameritox Ltd., in Greens-boro, North Carolina.
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24 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Ryan Collins and
Shane Needham
HPLC Column Technology in a Bioanalytical Contract Research Organization
High performance liquid chromatography–tandem mass spectrometry
(HPLC–MS-MS) is the go-to technique for high-throughput analysis of small-
molecule therapeutics, metabolites, and biomarkers. Through technological
advancements in the last decade, developing quality methods for a novel
analyte in the contract research environment has become easier and faster
than ever. Increasingly shorter run times, higher sensitivity, and greater
separation have all become possible in a standard method. This is, in part,
because of column technologies that have enabled the standardization of
the method development process. Method efficiency and productivity are
also improving because of emerging column technologies such as sub-2-μm
particles coupled with ultrahigh-pressure liquid chromatography (UHPLC)–
MS-MS, superficially porous particle columns, and microflow HPLC–MS-MS.
Increasing efficiency and productivity in high-throughput bioanalysis is
becoming more important as the applications for HPLC–MS-MS expand
to large molecules such as peptides, proteins, and oligonucleotides.
Over the course of the last several
decades, high performance liquid
chromatography–tandem mass
spectrometry (HPLC–MS-MS) has become
the method of choice for high-throughput
analysis of small-molecule therapeutics,
metabolites, and biomarkers. This is due,
in large part, to the selectivity and sensitiv-
ity provided by HPLC–MS-MS, combined
with the ability to rapidly develop an assay
consisting of quick extractions and short run
times for a vast majority of small molecules.
When presented with a new analyte at
a bioanalytical contract research organiza-
tion (CRO), the goal is to develop a robust
method with good chromatographic reso-
lution, repeatable results, and a quick run
time. However, after these scientific crite-
ria have been met, the ultimate end goal
for any bioanalytical CRO is productivity
and efficiency—analyzing the most sam-
ples possible while using the minimum
amount of solvent, supplies, and resources,
and still remaining scientifically sound.
In short, the goal at a CRO is to create
the most productive method in the most
efficient manner possible, all while using
sound science. This approach benefits not
only the CRO, but also its bioanalytical
clients, and most importantly, the end
users; patients that can receive care from
these novel therapeutics provided by the
industry.
There is an increasing trend in the
industry to monitor (possibly multiple)
metabolites, as well as a push toward using
HPLC–MS-MS for the analysis of large
molecules, including peptides and proteins.
As the industry shifts toward the analysis
of more-complicated therapeutics, there is
a need to increase efficiency and productiv-
ity wherever possible. With that in mind,
when developing a new HPLC–MS-MS
method for a novel molecule, every tool
in the chromatographic arsenal should be
used to grant the best chance of success.
Perhaps the strongest, most versatile tool in
the bioanalytical setting is the LC column.
A large reason that method development
can be performed with the amount of effi-
O
-
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 25www.chromatographyonline.com
ciency necessary to function as a CRO in
today’s bioanalytical world is the develop-
ment of column technology over the past
few decades. The reliable repeatability of
columns on the market today, combined
with the plethora of unique column types
that can be implemented, allow for the effi-
cient development of an HPLC–MS-MS
method for high-throughput analysis.
Because all bioanalytical work depends
on high-throughput analysis, many of the
trends in emerging technologies in the
bioanalytical market are directly related to
increasing on-instrument productivity and
reducing costs. This includes smaller par-
ticle size in columns coupled with ultrahigh
pressure liquid chromatography (UHPLC),
superficially porous shell column technol-
ogy, and microflow HPLC. This article
presents a quick background into the details
of developing an HPLC–MS-MS method
from the perspective of a CRO in relation
to column choice. It also focuses on recent
column technologies, the instrumentation
surrounding them, and their benefits in a
CRO environment.
Method Development
High-throughput bioanalysis CROs are usu-
ally a fast-paced environment, where it is nec-
essary to create a productive, rugged method
from the ground up for what is often times
an unknown novel therapeutic. A large part
of a CRO’s efficiency stems from its ability
to quickly develop a rugged method that
will repeatedly hold up to rigorous indus-
try and regulatory standards. As efficiency
can often be derived from simplicity, when
developing a new method the simplest solu-
tion is always the first approach. This is why,
despite the plethora of columns available for
use, it is almost always best to start with a
C18 or C8 column. One of the most versatile
and widely used columns, the C18 column
has been in use in one form or another for
decades. Comprising a simple octadecyl
carbon chain bonded silica-based stationary
phase, the C18 column is the go-to column
of choice for a large majority of molecules
analyzed by HPLC–MS-MS. C18 columns
have proven to provide good retention and
resolution for a vast array of small molecules.
With a proven track record of negligible
lot-to-lot and column-to-column variabil-
ity, there is minimal concern of anomalous
behavior throughout the life of a method
on a C18. C18 columns also tend to be very
rugged, with the average lifespan lasting for
upwards of thousands of injections. This is
a very important point in the development
of any method; if a seemingly scientifically
sound method has been developed, but the
column only lasts a few hundred injections
before peak deterioration, then the method
probably isn’t rugged or productive enough
to be feasible. A large benefit in the flex-
ibility of the C18 is that it allows for the
standardization of many HPLC–MS-MS
methods, which greatly increases the pro-
ductivity of high-throughput analysis.
With multiple standardized methods rely-
ing on one type of column and identical
mobile phases for an array of molecules,
it is possible to keep instruments running
continuously without interruption. This is
crucial to the high-volume requirement in
the bioanalytical CRO world.
However, there are always going to be
analytes that do not work on a C18 column.
For multiple analytes, resolution (Rs) and
chromatographic selectivity (α) will play
a role. However, here we focus on method
development of one analyte. Whether due
to poor retention (tR), poor asymmetry fac-
tor (AF), or poor repeatability, decisions
Is it a chiral molecule?
Is it a mobile phasemismatch?
Polarendcapped
column
Look at the functional groups andselect specialty column
Is it a mobile phasemismatch?
Chiral column
No
C18
Yes
No No
F5
C18
Ion pairing HILIC
Yes Yes
NonpolarPolar
Good tR
and good AF Good tR
and poor AFPoor tR
and good AF
Key
tR
= Retention time
AF = Asymmetry Factor
Poor tR
and poor AF
2-μm solid core
0.5-μm shell (3 μm total) 3-μm fully porous particle
Figure 1: Representative column method development flowchart.
Figure 2: Representative structure of SPP and fully porous particles.
-
26 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
can then be made on what type of specialty
column to look at. This process can quickly
become overwhelming given the plethora of
columns and column types on the market
today. Having an approach to address the
most common column-based issues during
method development, as seen in the flow-
chart in Figure 1, is an important aspect
in maintaining efficiency during method
development. Once it has become apparent
that a method will not be adequately devel-
oped on a C18 column, the next step is typi-
cally to evaluate the polar moieties and func-
tional groups exhibited by the molecule. For
a polar molecule, some of the more common
approaches available are to choose a polar
endcapped column or to implement an ion-
pairing reagent (where an ion-pairing reagent
such as heptafluorobutyric acid [HFBA] is
added to the mobile phases or extraction
solvents). When presented with a particu-
larly small, polar molecule, another option
available is to choose a column such as an F5
column (a pentafluorophenylpropyl station-
ary phase) or to use a hydrophilic-interaction
chromatography (HILIC) method. HILIC
methods use gradients with a high percent-
age of organic content coupled either with
an unmodified silica column, an amino col-
umn, a zwitterionic column, or any one of
a number of columns made specifically for
HILIC methods.
Recent Column Advancements
Although efficiency in method development
is paramount to being cost effective in a bio-
analytical CRO environment, this efficiency
would amount to nothing if the actual
methods themselves were not productive in
the long run. Even if all the scientific bench-
marks may have been met during develop-
ment, the overall costs of performing the
method determine whether it will actually
be feasible. The costs of a method are largely
determined based on two factors: the over-
all costs of disposable s