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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