TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2018 Waters Corporation 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 hypercalcemia 1 . Its amino acid sequence and structure are highlighted in Figure 1 2 . 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 operate 3,4 , 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 C 18 +, 90Å, 1.6 μm, 2.1 x 50 mm Column temperature: 60 o C; Sample temperature: 5 o C Injection volume: 20 µL Mobile phases: A: 0.1% Formic acid in H 2 O;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 C 18 , ACQUITY UPLC Peptide CSH C 18 , and CORTECS UPLC C 18 +) 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 C 18 +, 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 C 18 + Column, with its low level positive surface charge, provided significantly narrower peak widths (<4 seconds) than the traditional C 18 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 C 18 + 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% NH 4 OH 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 Time 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 % 0 100 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 87396.75 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 % Recovery % Matrix Effects % Recovery % Matrix Effects Time 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 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 Time 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 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 Time 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 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 MRM Calibration Curve (pg/mL) Weighting Linear Fit (R 2 ) % 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 C 18 +, 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