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INTRODUCTION
Salmon calcitonin (sCT) is a 32 amino acid (MW 3439.1 Da) synthetic
polypeptide that is used in the treatment of Paget’s disease,
osteoporosis, and hypercalcemia1. Its amino acid sequence and
structure are highlighted in Figure 12. Pharmacokinetics of sCT are
characterized by rapid absorption within 30 minutes and rapid
elimination with a half-life of ~ 1 hour, resulting in very low circulating
plasma levels (pg/mL)2,3
.
Although peptide biologics like sCT have historically been quantified
using ligand binding assays (LBAs), over the past few years there has
been a growing trend towards the bioanalysis of large molecules by LC-
MS. This is, in part, driven by the fact that LBAs can suffer from cross-
reactivity, lack of specificity, limited dynamic range, and long method
development times. In contrast, LC-MS has the advantage of shorter
development times, greater accuracy and precision, the ability to
multiplex, and can readily distinguish between closely related
analogues, metabolites, or endogenous interferences. Tandem
quadrupole instruments are the gold standard for routine LC-MS/MS
quantification of small molecules, peptides and digested proteins. As the
need to quantify intact larger peptides and proteins increases, HRMS
instruments are increasingly becoming an attractive orthogonal platform
for quantitative laboratories.
Previously described LC-MS methods for sCT have achieved LLOQ of
50 pg/mL by tuning the tandem quadrupole to achieve a resolution of
0.2 Da, as opposed to the unit resolution at which these instruments
usually operate3,4
, while other methods have used larger sample
volumes (500 µL) to achieve similar LLOQ’s. The work described here
uses UPLC separation, tandem quadrupole MS and simple, fast, and
selective sample preparation in a 96-well format to achieve a lower limit
of quantification (LLOQ) of 25 pg/mL, extracted from 100 µL of serum.
Additionally, the quantitative performance characteristics of a HRMS
system were evaluated for sCT and were found to be comparable to that
of a tandem quadrupole instrument.
HIGH SENSITIVITY QUANTIFICATION OF SALMON CALCITONIN FROM HUMAN SERUM USING MIXED-MODE SPE & LC-MS/MS
Nikunj Tanna & Mary Lame Waters Corporation, Milford, MA
Figure 1. Amino acid sequence and structure for salmon Calcitonin
(sCT)
METHODS
Sample Preparation
LC-MS/MS Conditions
LC system: ACQUITY UPLC I-Class Detection: Xevo TQ-XS Tandem Quadrupole Mass Spectrometer, ESI+ Column: CORTECS UPLC C18+, 90Å, 1.6 μm, 2.1 x 50 mm Column temperature: 60
oC; Sample temperature: 5
oC
Injection volume: 20 µL Mobile phases: A: 0.1% Formic acid in H2O;B: 0.1% Formic acid in ACN
LC Gradient:
MS conditions: MRM Transitions:
Data management: Instrument control software: MassLynx (v4.2) Quantification software: TargetLynx
References
1. Zeng K., Geerlof-Vidavisky I., Gucinski A., Jiang X., and BoyneII M. Liquid Chromatography-High
Resolution Mass Spectrometry for Peptide Drug Quality Control. The AAPS Journal, Vol. 17, No. 3,
2015 (# 2015).
2. Novartis (Oct 2009) Miacalcin (Calcitonin-salmon) Unites States drug package insert.
3. Salmon calcitonin, structure from Drugbank. Retrieved 30May2018 from https://www.drugbank.ca/
drugs/DB00017
4. Li Y., Hackman M., and Wang C. Quantitation of Polypeptides (Glucagon and Salmon Calcitonin) in
Plasma Samples by ‘High Resolution’ on a Triple Quadrupole Mass Spectrometer. Bioanalysis (2012) 4
(6), 685–691.
RESULTS & DISCUSSION
Sample Preparation
Protein precipitation with high volumes of organic solvent is usually the preferred sample preparation strategy used for most small molecule analysis. However, this strategy does not always work for peptides and proteins due to lack of solubility of these analytes in high organic solvents. Solid phase extraction (SPE) is usually the preferred extraction technique for peptides/proteins. In the case of sCT, different sample:protein precipitation solvent ratios and different SPE sorbent materials (based on Waters Oasis PST SPE method development protocol) were evaluated (data not shown). Protein precipitation with 1:1 (v;v) acetonitrile, followed by SPE using Oasis WCX 96-well µelution format gave % recovery >90% and % matrix effects <15% as shown in Figure 2
Liquid Chromatography
Several reversed-phase columns (ACQUITY UPLC Peptide HSS T3, ACQUITY UPLC Peptide BEH C18, ACQUITY UPLC Peptide CSH C18, and CORTECS UPLC C18+) were evaluated for overall chromatographic performance (e.g., assessment of peak shape, area counts and signal to noise). Best chromatographic separation was achieved using a CORTECS UPLC C18+, 90Å, 1.6 μm, 2.1 x 50 mm column and 0.1% formic acid in water and acetonitrile mobile phases. Unlike small molecules, larger peptides and small proteins, like sCT, suffer from poor mass transfer in and out of fully-porous particles. Thus, use of a sub-2 µm solid-core CORTECS UPLC C18+ Column, with its low level positive surface charge, provided significantly narrower peak widths (<4 seconds) than the traditional C18 column (>12 seconds) and resulted in 10-fold improvement in S/N (Figure 3).
Tandem Quadrupole
A MS Full scan experiment performed on the Xevo TQ-XS system showed several multiply charged precursors (data not shown). Of these, the 4+ (m/z 859.2) and 5+ (m/z 687.5) precursors were the most predominant species and yielded highly selective fragments which were used for quantification. The MRM transition using 687.5 > 830.3 was used as the primary quantification transition, while the MRM transition 859.2 > 1106.7 was used as a qualifier.
Using only 100 µL of human serum and the selective sample cleanup method described above, this method achieved a LLOQ of 25 pg/mL for sCT. Using a 1/x linear fit, calibration curves were linear (r
2 >0.99) from 25-1,500 pg/mL with accuracies between 85-115 % and CVs <15% for all points on
the curve (Table 1). Additionally, both intra-day and inter-day accuracy and precision met recommended bioanalytical method validation guidelines. This intra and inter day QC performance is highlighted in Table 2, while chromatographic performance is illustrated in Figure 4.
HRMS
The quantitative performance of the Xevo G2-XS QTof MS was evaluated with focus on the 4+ (859.146 m/z) and 5+ (687.911 m/z) precursors for salmon calcitonin. The different acquisition modes available on the HRMS instrument, Full Scan, Tof MRM (Precursor>Precursor) with Target Enhancement (TE), and Tof MRM (Precursor>Product) with Target Enhancement (TE) were evaluated (data not shown).
Best overall quantification was achieved using the targeted Tof MRM (Precursor> Precursor) mode with TE, using very low collision energy for the 4+ and 5+ precursors of salmon calcitonin. This performance was comparable to the quantification data observed on the Xevo TQ-XS. A summary of standard curve performance for the 4+ and 5+ precursors of salmon calcitonin for both the Xevo G2-XS QTof and Xevo TQ-XS is highlighted in Table 1. Using the Xevo G2-XS QTof and the targeted Tof MRM (Precursor>Precursor) method, the lower limit of quantification (LLOQ) achieved was 50 pg/mL with a dynamic range from 50-1,500 pg/mL. The calibration curve was linear with r
2 values >0.99 (1/x weighting) with mean accuracy of all calibration
points between 85-105% and 85-110% for the 4+ and 5+ precursors, respectively (Table 1). Quality Control (QC) performance of salmon calcitonin was excellent, with accuracies between 93-108 % and CV’s < 11% for both the 4+ and 5+ precursors, respectively (Table 3). QC chromatographic performance is highlighted in Figure 5 for 4+ (panel A) and 5+ (panel B) precursors.
CONCLUSION
It is critical to evaluate and optimize every step of the analysis workflow to achieve low pg/mL sensitivities for peptides/proteins. In the case of sCT, we found;
Protein precipitation with a low volume of organic solvent, followed by selective SPE using Oasis WCX µelution SPE gave recoveries of 90% and matrix effects <15% for sCT.
The low level positive surface charge and sub-2 µm solid core of CORTECS C18+ column gave the best chromatographic performance for sCT with peak width ≈4 seconds and S/N improvement >10x
A highly sensitive Xevo TQ-XS system achieved a LLOQ of 25 pg/mL and linearity from 25-1500 pg/mL with all calibration curve and QC points within the bioanalytical acceptance criteria using only 100 µL of serum.
Quantitative performance of the Xevo G2-XS was similar to the Xevo TQ-XS, thus providing scientists with a single flexible platform which can be used for both qualitative and quantitative experiments.
Pretreatment: 100 µL sample + 100 µL 100% Acetonitrile
Dilution: 150 µL supernatant + 600 µL 4% Phosphoric acid
Load 2x375 µL from above onto a Oasis® WCX µElution plate
Wash 1: 200 µL 5% NH4OH
Wash 2: 200 µL 20% Acetonitrile
Elute 25 µL 1% TFA in 75/25 Acetonitrile/Water
Dilute with 25 µL 100% Water
Time (min) Flow rate (mL/min)
% A % B Curve
0.0 0.4 95 5 6
0.5 0.4 95 5 6
2.5 0.4 25 75 6
2.6 0.4 5 95 6
3.5 0.4 5 95 6
3.6 0.4 95 5 6
4.5 0.4 95 5 6
Capillary: 1.0 kV
Source offset : 30 V
Source temp: 150 °C
Desolvation temp.: 400 °C
Cone gas flow: 150 L/Hr
Desolvation gas flow: 900 L/Hr
Collision gas flow: 0.15 mL/Min
Nebulizer gas flow: 7 Bar
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40
%
0
10087396.75
88706.58
40515.22
77042.15
HSS T3, 2.1x50 mm, 1.8µm
Peak width =~ 6 seconds
S/N = 900.25
Peak Area = 87396.75
Peptide BEH C18, 2.1x50 mm,
Peak width = ~ 12 seconds
S/N= 386.06
Peak Area = 88706.58
Peptide CSH C18, 2.1x50 mm
Peak width = 4 seconds
S/N= 259.15
Peak Area = 40515.22
CORTECS C18+, 2.1x50 mm, 1.6µm*
Peak width = 4 seconds
S/N= 2240.66
Peak Area = 77042.15
Ultimately chosen for quantification
90.6
-13.9-20.00
0.00
20.00
40.00
60.00
80.00
100.00
Without Conditioning
% R
eco
very
% M
atri
x Ef
fect
s
% Recovery % Matrix Effects
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
%
0
100
631.54
877.75
1326.98
4389.18
Blank
LLOQ-50 pg/mL
LQC-100 pg/mL
MQC-500 pg/mL
HQC-1000 pg/mL
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80
%
0
100
67.77
198.31
290.18
728.73
Blank
LLOQ-50 pg/mL
LQC-100 pg/mL
MQC-500 pg/mL
HQC-1000 pg/mL
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
Transition 687.3830.3
Blank
LLOQ-25 pg/mL
LQC-75 pg/mL
MQC-250 pg/mL
HQC-1000 pg/mL
Instrument Precursor MRMCalibration
Curve(pg/mL)
WeightingLinear Fit
(R2)% Accuracy
Range
Xevo TQ-XS 4+ 687.3>830.3 25-1,500 1/x >0.99 85-115
Xevo G2-XS Tof 4+ 687.911>687.911 50-1,500 1/x >0.99 85-105
Xevo TQ-XS 5+ 859.2>1106.7 25-1,500 1/x >0.99 86-112
Xevo G2-XS Tof 5+ 859.146>859.146 50-1,500 1/x >0.99 85-110
QC
Level
Expected
Concentration
(pg/mL)
Intra-Day Inter-Day
Average
Observed% CV % Recovery
Average
Observed% CV % Recovery
LQC 100 104.1 2.99 104.1 101.5 2.23 101.5
MQC 500 444.7 0.35 88.9 436.0 2.8 87.2
HQC 1000 968.4 9.53 96.8 866.8 4.97 86.7
QC
Level
Expected
Concentration
(pg/mL)
Intra-Day Inter-Day
Average
Observed% CV % Recovery
Average
Observed% CV % Recovery
LQC 75 77.2 12.8 97.1 75.71 11.2 99.1
MQC 250 240.6 8.84 103.8 242.2 8.32 103.1
HQC 1000 891.7 9.24 110.8 993.7 10.9 100.6
Figure 2. % Recovery and % Matrix effects for extraction of sCT from serum using protein precipitation followed by Oasis WCX SPE
Figure 3. Chromatographic performance of sCT on different C18 column chemistries. CORTECS UPLC C18+, 90Å was chosen for the assay
Figure 5 (B). Representative chromatograms for Tof MRM (precursor > precursor) for precursor 5+ (859.146) of sCT on Xevo G2-XS
Figure 5 (A). Representative chromatograms for Tof MRM (precursor > precursor) for precursor 4+ (687.911) of sCT on Xevo G2-XS
Figure 4. Representative chromatograms for MRM transition 687.5 > 830.3 of sCT on Xevo TQ-XS
Table 2. Representative Intra and Inter day QC statistics for sCT on Xevo TQ-XS
Table 1. Calibration curve statistics for sCT on Xevo TQ-XS and Xevo G2-XS
Table 3. Representative Intra and Inter day QC statistics for sCT on Xevo G2-XS
Xevo TQ-XS
Xevo G2-XS
Xevo G2-XS