Intrada Amino Acid Separates Intact Amino Acids in Human Serum

52 Pages·2014·8.28 MB·English

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June 2014
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TABLE OF CONTENTS
THE APPLICATION

NOTEBOOK
Medical/Biological
8 Utilization of CESI Technology for
Comprehensive Characterization of Biologics
Rajeswari Lakshmanan, PhD, AB Sciex
10 High Resolution LC–MS Analysis of Reduced IgG1
Monoclonal Antibody Fragments Using HALO Protein C4
Stephanie A. Schuster and Barry E. Boyes , Advanced Materials Technology, Inc.
11 Intrada Amino Acid Separates
Intact Amino Acids in Human Serum
Piotr Macech, Robert Puryear, and Itaru Yazawa, Imtakt USA
12 Separation of Nucleobases Using
TSKgel® SuperSW mAb HTP Column in HILIC Mode
Justin Steve and Atis Chakrabarti, PhD, Tosoh Bioscience LLC
14 Characterization of Tobacco Extracts by
Gas Chromatography High Resolution
Time of Flight Mass Spectrometry (GC-HRT)
David E. Alonso, Jonathan Byer, Jeff Patrick, and Joe Binkley, Life Science
and Chemical Analysis Centre, LECO Corporation
Chiral
15 Isolation of Structurally Related Components in Natural
Product Extracts Using the RegisPack and Whelk-O1 Chiral
Selectors
Andrew Geyer and Ted Szczerba, Regis Technologies, Inc.
THE APPLICATION NOTEBOOK – JUNE 2014 3
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TABLE OF CONTENTS
Environmental
16 Keeping Water Safe: Detecting Pharmaceutical
and Personal Care Products in Water Using Liquid
Chromatography–Mass Spectrometry
Joe Anacleto, Zicheng Yang, Helen (Qingyu) Sun, and Kefei Wang, Bruker
Daltonics
18 Solid Phase Extraction of Organophosphorus
Pesticides and Triazine Herbicides in Water
Using a New Polymeric Sorbent
Xiaoyan Wang, UCT, LLC
Food and Beverage
19 Determination of Phenolic Compounds in Virgin Olive Oil
Using Comprehensive 2D-LC
Sonja Krieger and Sonja Schneider, Agilent Technologies Inc.
20 The Analysis of Chlorinated Dioxins, Difurans, and
Polychlorinated Biphenyls in Edible Oils
FMS, Inc.
21 Structural Differences in Modified Starches
Malvern Instruments Ltd.
22 Detection of Low-Level Sulfur Compounds in
Spearmint Oil Using the Pulsed Flame Photometric
Detector (PFPD)
Gary Engelhart and Cynthia Elmore, OI Analytical
23 HILIC with Increased Sensitivity for the Analysis of
Sugars
Melissa Turcotte and Satoko Sakai, Showa Denko America and Showa Denko
K.K.
24 Veterinary Drug Residue Analysis Using the
AutoMate-Q40: An Automated Solution to QuEChERS
Tyler Trent, Teledyne Tekmar
4 THE APPLICATION NOTEBOOK – JUNE 2014
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TABLE OF CONTENTS
Pharmaceutical/Drug Discovery
25 Fast Screening Methods for Analgesics and Non-Steroidal
Anti-Inflammatory (NSAIDS) Drugs by HPLC with Agilent
Poroshell 120 Columns
William Long, Agilent Technologies, Inc.
28 Separation of Five Steroids on a C18-Functionalized
Polymeric Reversed-Phase HPLC Column (PRP™-C18)
Derek Jensen and Mark Carrier, Hamilton Company
29 Measuring Antibody Molecular Weight by SEC-MALS
Malvern Instruments Ltd.
30 Accurate Pain Management Analysis in
Under 5 Min on Raptor™ Biphenyl
Superficially Porous Particle LC Columns
Sharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation
32 Analysis of Alprostadil by HPLC
with Post-Column Derivatization
Pickering Laboratories, Inc.
33 Antibody Drug Conjugate (ADC) Analysis
Wyatt Technology
34 Characterization of PLGA Using SEC–MALS-VIS
Wyatt Technology
General
35 Separation of a Mix of Acidic, Basic, and Neutral
Compounds at High pH Conditions
Diamond Analytics
36 Concentrating Phenolic Compounds Using
the XcelVap® Concentration System
David Gallagher, Horizon Technology, Inc.
37 Recent Improvements in Time of Flight
Mass Spectrometer Detection Technologies
PHOTONIS USA
THE APPLICATION NOTEBOOK Ð JUNE 2014 5
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TABLE OF CONTENTS
Articles
38 The 2014 LCGC Awards
Meg L’Heureux
43 Recent Progress in Chiral Stationary Phase Development
and Current Chiral Applications
Timothy J. Ward and Karen D. Ward
47 The Fundamental Shift to Tandem Mass Spectrometry
St. John Skilton, Eric Johansen, and Xu Guo
Departments
51 Call for Application Notes
Cover Photography: Getty Images
6 THE APPLICATION NOTEBOOK – JUNE 2014
mbcyyelaaallcgonkewnta ES445794_LCGCSUPP0614_006.pgs 05.28.2014 00:06 ADV
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THE APPLICATION NOTEBOOK – JUNE 2014 7
mbcyyelaaallcgonkewnta ES443904_LCGCSUPP0614_007.pgs 05.22.2014 23:27 ADV
MEDICAL/BIOLOGICAL
Utilization of CESI Technology
Experimental Conditions
Trastuzumab was reduced, alkylated, and digested with trypsin.
for Comprehensive
After drying, it was resuspended in the leading electrolyte (100
Characterization of Biologics mM ammonium acetate at pH 4) and 50 nL (100 fmol) was
injected into the separation capillary. The background elec-
Rajeswari Lakshmanan, PhD, AB Sciex trolyte used was 10% acetic acid and a separation voltage of
20 kV (normal polarity) was applied for 60 min. Information
Monoclonal antibodies (mAbs) form a major class of biologics and dependent acquisition (IDA) was utilized to trigger MS-MS. IDA
recently biosimilars and biobetters are being added to the grow- parameters were optimized so that the duty cycle of the MS
ing inventory of therapeutics. In-depth characterization of mAbs at was matched to the high speed CE separation. Data analysis
various stages of development and manufacturing is essential to was performed using BioPharmaView™ software (AB Sciex,
maintain product safety and eff cacy. However, analysis of mAbs is Massachusetts).
challenging due to their high molecular weight, the microheteroge-
neity presented by the glycans, and degradative modif cations that Results and Discussion
occur during production. Any analytical technique that provides Primary Sequence Coverage: 100% primary sequence coverage of
greater depth of information without a time penalty is an advantage. both the light and heavy chains of the antibody were obtained. Pep-
A recent advancement to meet this need was the introduction of tides ranging from 4 to 63 amino acids in length without any missed
CESI–MS. CESI is the integration of capillary electrophoresis (CE) cleavages were detected. Electrophoretic separation is based on the
and electrospray ionization (ESI) in a dynamic process, within the charge-to-mass ratio of the peptides and is not dependent on rela-
same device. In this technology, the analytes are separated inside tive hydrophobicity. Thus, small hydrophilic peptides often lost in
an open nontapered capillary based on their electrophoretic mobil- the LC void volume, and large hydrophobic peptides, which tend to
ity, and electrosprayed directly into the MS (2). At operating f ow be retained on the column, can be identif ed by CESI–MS, resulting
rates less than 30 nL/min, very eff cient desolvation and, thus, ion- in the high sequence coverage observed.
ization is achieved.
Though high speed CESI separations reduce analysis time, it also PTM Characterization: Data from CESI-MS analysis showed the
necessitates the use of high speed MS to preserve the separation presence of several PTM hotspots such as N-terminal pyroGlu
eff ciency. The TripleTOF® 5600+ system (AB Sciex) offers the formation, methionine oxidation, and asparagine deamidation.
necessary high acquisition speed, high resolution, and high mass Pyroglutamination leads to loss of a positive charge which re-
accuracy, in both MS and MS-MS modes. CESI performance was sults in the electrophoretic mobility of the modif ed peptide being
evaluated by analyzing a tryptic digest of trastuzumab using the lower than the unmodif ed one. This is advantageous since the
SCIEX CESI 8000 - TripleTOF® 5600+ MS platform. modif ed and unmodif ed forms can be separated by CESI–MS
and the MS-MS spectra conf rmed the presence of the pyroGlu-
tamate residue (Figure 1). Oxidative degrada-
tions at Met255 and Met431 and deamidations at
Asn55 and Asn387 in the heavy chain and at Asn30
4.0e6 N-terminal Glu (a) in the light chain were also identif ed. A typical
3.5e6
3.0e6 identif cation from the CESI–MS data is shown
Intensity 221...505eee666 in Figure 2.
1.0e6
5.0e5 N-terminal pyroGlu
SPpreeccutrrusomr: 0 f9r.o30m2e.0 56 DDeac13Her2u2g9P.e5rul_100mMLE_3205.00TOF501Db*A3333093io.01n6.s559s8ec500cps_1t3o15.W0ithChargeSel_321.w.5iff (sample 1) -3 H2e.r010, Experiment3 120.5, +TOF MS^2 (3130T.0i0m - e2 5(0m0i)n f)rom 3353.3.522 min 34.0 34.5 35.0 35.5 36.0 36.5 (b37).0 Glycosylation Heterogeneity: Trastuzumab pos-
6500 sesses one N-glycosylation site at Asn300 in
6000 the HC where different glycoforms such as a-
5500
fucosylated or fucosylated glycans can be pres-
5000
y
4500 *586.36339 ent (3). By using CESI-MS, the G0F, G1F, and
4000 b
Intensity 33500000 *452.24540 Gw2erFe fsoerpmasra toefd twheel l paesp stihdoew nT KinP RFEigEuQreY N3S. TFYuRr-
2500 b5 *1y31134.6853 thermore, the identification of G0F, G1F, and
21050000 *551.3233 *71y4.73923 y y *11y841.63458 G2F forms of peptide EEQYNSTYR without the
1050000 b2 *424.2592 *6b806.3647 b7*813.84611 b9*9b38y1.409616 *98y31.50663*1y0410.15b85121*10971.62123 *1314.6921*1y41125.7647 pmeipsstiedde cTleKaPvRaEgeE Q(aYtN tShTeY aRrg) inalisnoe creosnifdirume eidn tthhee
0150 200 250 300 350 400 450 500 550 600 650 700 750m/z800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 presence of these glycoforms. In addition, the
Figure 1: (a) Extracted ion electropherogram of N-terminal peptide with Glu and pyroGlu a-fucosylated forms of this peptide, such as G0
separated by CESI and (b) MS-MS identif cation of N-terminal peptide with pyroGlu. and G1, were identified, but it has to be further
8 THE APPLICATION NOTEBOOK – JUNE 2014
mbcyyelaaallcgonkewnta ES446237_LCGCSUPP0614_008.pgs 05.28.2014 18:40 ADV
MEDICAL/BIOLOGICAL
separation eff ciency and high sensitivity allows the
SPpreeccutrr4uso.m5r: e f5r4o4m2. 86 DDeac13HUer2ugnPeruml_100omMLdE_25i0fTOF5e01DdA30i onps5seec50p0cpst_NiOCdhargeeSel_2.wiff (sample 1) - Her8, Experiment 19, +TOF MS^2 (100 - 250N0) from 27.027 min T P y7*808.3885 (1) Y (a) aanbaulnydsiasn ot f saplle pceiepst,i diens aindcdluitidoinn gt om ocdoinf fe rdm ainngd ltohwe
4.0e4
3.5e4 *404.7045 (2) amino acid sequence of the antibody.
3.0e4 *249.1643 y
Intensity 22..50ee44 *136.0803 b *610y.29527 *711.36387 (1) References
1.5e4 *277.15295 (1) 809.3908 (1) (1) J.M. Busnel, B. Schoenmaker, R. Ramautar, A.
150...000eee430 150*171.1y18112*01099.1135 250 278.3105083 (1) 350 400405.2075 *(244)8560.2354*4y9654.025034m/z550 600*611.2996550 700712.33907 (510)*790.3809 8(10)0 850 900 959071y.84573 Cm94aar7nr6a, –sAc9.o4 -D8P3eae n(l2cdo0er1rb,0 oa),.n Cd. OR.a tMnaayyabkoer,o dJ.a F, eAitnealsl.o nC,h Je.m C.h8ap2-,
SPpreeccutrrusomr: f5ro4m3. 36 DDeac13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_NOChargeSel_2.wiff (sample 1) - Her8, Experiment 9, +TOF MS^2 (100 - 2500) from 28.180 min y (b)
3.0e4 Deamidated at Asn55 *405.1974 N* T P *8709.3766 Y (2) A. Beck, S. Sanglier-Cianferani, and A. Van Dorsselaer,
Anal. Chem.84, 4637–4646 (2012).
2.5e4
Intensity21..05ee44 *136.0805 *249.1646 *49y6.42536 *61y1.25788 (1) *7y162.3253
1.0e4
b
5.0e3 *171.1y1717 *277.21594 *405.7015 *486.7298 612.2888 (1) *722.3113 *810.3828 971y.48457
0.0e0 150 200 250 300 350 400 450 500 m/z550 600 650 700 750 800 850 900 950
Figure 2: MS-MS identif cation of asparagine deamidation with (a) showing unmodif ed
peptide and (b) deamidation at Asn55 in the heavy chain of trastuzumab.
XXXIIICCC fffrrrooommm 00 099922299922002110331__3TT_rrTaarssa11smm1ggmmmgLLm__11L00_001mm00MMmLLEEM__LRRE77_..wwR7iiff.ffw((ssaaifmmf(ppsllaeem11))p--00le99122)99-220009112339__2TT0rraa1ss311_mmTrggammsLL1__m11g00m00mmLMM_1LL0EE0__mRR77M,, EELxxEpp_eeRrr7iimm, eeEnnxttp11e,, r++imTTOOeFFn tMM1SS, +((11T00O00F--22 M0000S00 ())1:: 10101094-2370..78082000)+0: +/1-/00-.030.920.528D50Da0a+/-0.025Da
5.2e5 G0F 25.42
5.0e5
4.8e5
4.6e5
4.4e5
4.2e5
4.0e5
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
Intensity 222...864eee555
2.2e5
2.0e5
1.8e5 G1F
1.6e5
1.4e5 25.7
1.2e5
1.0e5
8.0e4 G2F
6.0e4
4.0e4 25.95
20..00ee40 24.8 24.9 25.0 2255.1.10 25.2 25.3 25.4 25.5 25.6Time (min2)5.7 25.8 25.9 26.0 26.1 26.2 26.3 26.4 26.5
Figure 3: Extracted ion electropherograms of peptide TKPREEQYNSTYR with G0F, G1F,
and G2F modif cations.
confirmed that the a-fucosylated forms were not generated due
to source fragmentation of fucosylated counterparts.
Conclusions
We have presented CESI–MS, a robust ultra-low f ow and highly
eff cient separation technology in combination with TripleTOF MS,
AB Sciex
a high resolution accurate mass measurement system for qualita-
tive analysis of biopharmaceuticals. CESI–MS is attractive for simul- 500 Old Connecticut Path, Framingham, MA 01701
taneous analysis of primary sequence coverage and glycopeptide tel. [phone], fax [phone]
prof ling, without carry-over concerns. The combination of high Website:www.sciex.com/cesi
THE APPLICATION NOTEBOOK – JUNE 2014 9
mbcyyelaaallcgonkewnta ES446241_LCGCSUPP0614_009.pgs 05.28.2014 18:41 ADV
MEDICAL/BIOLOGICAL
High Resolution LC–MS Analysis of Reduced IgG1 Monoclonal
Antibody Fragments Using HALO Protein C4
Stephanie A. Schuster and Barry E. Boyes, Advanced Materials Technology, Inc.
A new fused-core particle designed specif cally for the sepa- propanol/0.5% (v/v) formic acid with 20 mM ammonium formate;
ration of proteins and monoclonal antibodies with 3.4 µm temperature: 80 °C; f ow rate: 0.4 mL/min; instrument: Shimadzu
particle size and a thin 0.2 µm porous shell demonstrates high LCMS 2020; detection: 280 nm; MS-2020 single quadrupole MS in-
resolution of the heavy and light chain fragments of IgG1. strument using ESI at +4.5 kV capillary voltage, 2 pps scan rate from
500 to 2000 amu m/z. Component masses for these measurements
The focus of many pharmaceutical companies has shifted to larger were determined by deconvolution using MagTran software, v. 1.02,
biotherapeutic molecules as potential treatments for disease. These based on the ZScore algorithm developed by Zhang and Marshall (1).
biomolecules present a new set of challenges compared to their
small molecule predecessors when complete characterization is Results
considered. Variants can be found through different glycans, de- The addition of a small amount of ammonium formate to formic acid
amidations, enzymatic clipped polypeptides, and so on, not to men- in the mobile phase improves the peak shapes. An optimized, shallow
tion the potential for aggregate formation. Rapid reversed-phase LC gradient allows resolution of the variants of both the heavy and light
analysis is convenient for its ability to be interfaced to online mass chain fragments of IgG1 as shown in Figure 1. The MS spectra can then
spectrometry. The HALO Protein C4 bonded phase is ideal for this be deconvoluted to determine the masses associated with each peak.
application with its optimized design for high recovery and stability
at the high temperatures and low pH required by the analysis. Conclusions
HALO Protein C4 demonstrates the low pH and high temperature
Experimental Conditions stability that is required to analyze reduced and alkylated IgG1 us-
Column: 100 mm × 2.1 mm Halo Protein C4; gradient: 29–32% ing mass-spec friendly mobile phase. The use of 80 °C enables
B in 20 min; mobile phase A: 0.5% (v/v) formic acid with 20 mM improved peak shape while the high resolution allows complete
ammonium formate; mobile phase B: 45% acetonitrile, 45% iso- analysis of the IgG1 fragments that are present.
References
(1) Z. Zhang and A.G. Marshall, J. Am. Soc. Mass Spectrom.9, 225 (1998).



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Figure 1: LC–MS separation of reduced IgG1. Column: 2.1 mm i.d ×
100 mm HALO Protein C4; Flow rate: 0.4 mL/min. Mobile phase A: 0.5 MAC-MOD Analytical, Inc.
% formic acid with 20 mM ammonium formate; mobile phase B: 45%
103 Commons Court, Chadds Ford, PA 19317
AcN/45% IPA/ 0.5 % formic acid with 20 mM ammonium formate; gradi-
tel. [phone]; fax [phone]
ent: 29–32% B in 20 min.; temperature: 80 °C; detection: 280 nm; MS
conditions: Shimadzu LCMS-2020, ESI +4.5 kV, 2 pps, 500–2000 m/z. Website: www.mac-mod.com
10 THE APPLICATION NOTEBOOK – JUNE 2014
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