Exploring LC-MS peptide dynamic rangeFrom enhanced base signal to the higher end of the dynamic range in 10log steps

Anne Kleinnijenhuisanne.kleinnijenhuis@triskelion.nl

Frédérique van HolthoonJan Toersche

Aim of the study

We observed that addition of a suitable internal standard can enhance the analyte signal at lower concentrations.

Usually the most suitable IS is a stable isotope labeled internal standard (SIL IS) due to the almost identical properties.

FTISADTSK (tryptic trastuzumab peptide, FTI) was analyzed using UPLC-MS/MS (triple quadrupole) in the absence and presence of FTISADTSK-13C6

15N2 (FTI IS) to investigate the effect of the presence of SIL IS on the analyte base signal.

In addition the extent of the dynamic range, ion ratios and absolute signals were investigated.

Experimental (1)Preparation of stock solutions and reagents

Diluent: 0.5% TFA, 1% FA, 10% ACN in MQ.

FTI 4 µg/ml stock: dissolve 1 nmol (99.2% purity) in 240 µl diluent.

FTI IS 4 µg/ml stock: dissolve 1 nmol (96.8% purity) in 236 µl diluent.

FTI IS diluent (4 ng/ml): dilute FTI IS 4 µg/ml stock 1000-fold with diluent.

Experimental (2)Preparation of calibration samplesCalibration sample Solution Volume

(µl) Solution Volume (µl)

End volume (µl)

FTI 100 ng/ml FTI 4 µg/ml 25 Diluent 975 1000FTI 10 ng/ml FTI 100 ng/ml 100 Diluent 900 1000FTI 1 ng/ml FTI 10 ng/ml 100 Diluent 900 1000FTI 100 pg/ml FTI 1 ng/ml 100 Diluent 900 1000FTI 10 pg/ml FTI 100 pg/ml 100 Diluent 900 1000FTI 1 pg/ml FTI 10 pg/ml 100 Diluent 900 1000FTI 100 fg/ml FTI 1 pg/ml 100 Diluent 900 1000FTI 0 fg/ml - - Diluent 200 200

Calibration sample Solution Volume (µl) Solution Volume

(µl)End volume

(µl)FTI 100 ng/ml + IS FTI 4 µg/ml 25 FTI IS diluent 975 1000FTI 10 ng/ml + IS FTI 100 ng/ml + IS 100 FTI IS diluent 900 1000FTI 1 ng/ml + IS FTI 10 ng/ml + IS 100 FTI IS diluent 900 1000FTI 100 pg/ml + IS FTI 1 ng/ml + IS 100 FTI IS diluent 900 1000FTI 10 pg/ml + IS FTI 100 pg/ml + IS 100 FTI IS diluent 900 1000FTI 1 pg/ml + IS FTI 10 pg/ml + IS 100 FTI IS diluent 900 1000FTI 100 fg/ml + IS FTI 1 pg/ml + IS 100 FTI IS diluent 900 1000FTI 0 fg/ml + IS - - FTI IS diluent 200 200

Experimental (3)

Parameter SettingUPLC Acquity (Waters)Column temperature (°C) 40 Column Acquity HSS T3, 100 x 2.1 mm, 1.8 µm (Waters)Flow rate (µl/min) 500Mobile phase A 0.1% FA in MQMobile phase B 0.1% FA in ACNInjection volume 10 µl (full loop)Mass spectrometer Xevo TQ-S (Waters)Ionization mode Electrospray ionization (positive mode)Source temperature (°C) 150Desolvation temperature (°C) 600Cone gas flow (L/h) 150Desolvation gas flow (L/h) 1200

UPLC and MS general parameters

Experimental (4)UPLC gradient

Time (min) Mobile phase A (%) Mobile phase B (%) Divert valve0 92 8 Waste

0.5 LC1 92 8

6.5 73 276.75 5 95

7 5 957.25 92 8

8 92 8 Waste

Experimental (5)MS/MS conditionsPeptide Precursor

ion (m/z)Product ion (m/z)

Dwell time (ms)

Cone(V)

Collision energy

(eV)

Assignment

FTISADTSK 485.25 721.4608.3221.1

525252

161616

121210

[M+2H]2+ => y7+

[M+2H]2+ => y6+

[M+2H]2+ => a2+

FTISADTSK-13C615N2

(SIL IS)489.25 729.4

616.3221.1

525252

161616

121210

[M+2H]2+ => y7+

[M+2H]2+ => y6+

[M+2H]2+ => a2+

Quantifiers are underlined.

FTI and FTI IS MS/MS settings were matched.

Results & Discussion (1)

Concentration FTI(pg/ml)

Mean peak area FTI quantifier (n=3) RSD in peak area (%)

FTI FTI+IS FTI FTI+IS

0 0 2 #DIV/0! 35

0.1 0 6 173 18

1 23 24 25 14

10 265 297 4.6 3.9

100 2740 2759 1.1 2.8

1000 27959 28417 1.5 1.1

10000 278818 283923 0.4 2.2

100000 2732291 2763342 0.4 2.8

Correlation coefficient r (linear regression, no weighting):

0.1-100000 0.99999792 0.99999625

1-100000 0.99999794 0.99999628

FTI result table (triplicate analyses)

Nearly perfect relation between

signal and FTI concentration

Results & Discussion (2)Calibration curve

1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

Calibration curve FTISADTSK

10log (concentration FTI in fg/ml)

10lo

g (m

ean

peak

are

a)

Results & Discussion (3)Chromatograms quantifier triplicates (blank)

Results & Discussion (4)Chromatograms quantifier triplicates (100 fg/ml)

Results & Discussion (5)Chromatograms quantifier triplicates (1 pg/ml)

Results & Discussion (6)Chromatograms quantifier triplicates (1 ng/ml)

Results & Discussion (7)General remarks

Dynamic range at least 6 orders of magnitude.

Enhanced FTI analyte base signal and higher precision at lower FTI concentrations after addition of FTI IS.

Linear behavior between 1 and 100000 pg/ml, r = 0.99999x.

The study was performed in pure solvents. It is assumed that the presence of similar compounds and/or matrix complexity could also result in enhanced base signal.

Enhanced base signal effect is attributed to the SIL IS attenuation of factors which deteriorate method performance for an analyte, e.g. adsorption.

Results & Discussion (8)Assessment signal saturation range

Signal saturation in Xevo TQ-S takes place at peak height 1.342 . 108

Peak height at 100 ng/ml for FTI quantifier around 7.3 . 107

=> Linear dynamic range probably extends to >100 ng/ml, possibly to approximately 180 ng/ml.

Close to and/or after signal saturation there is a non-linear relation between concentration and peak area. The signal increases as peak width only.

Results & Discussion (9)Assessment detection limit range

Base signal enhanced at 100 fg/ml after addition of FTI IS.

Enhancement effect might be more pronounced for analytes which have major analytical issues. FTI is relatively straightforward to analyze.

Below 1 pg/ml non-linear behavior of FTI signal.

However, signal still present at 100 fg/ml.

RSD in FTI peak area increases at lower concentrations.

Results & Discussion (10)Expanding the opposite extremes of the linear range

Solutions for expanding the linear range near the detection limit:Increase injection volumeIncrease dwell timeUse smaller scale LC / nanospray

Solutions for expanding the linear range near signal saturation:Decrease injection volumeDecrease dwell timeUse less intense transitionUse lower-abundance isotope as precursorDeoptimize parameters e.g. cone voltage or needle position. However, suboptimal settings could increase analytical variation.Apply saturation correction algorithm.

Results & Discussion (11)Quantifier and qualifier transitions FTI: 1.00 ng/ml FTI IS: 4.00 ng/ml

Results & Discussion (12)Ion ratios

Ion ratios are reproducible and similar between FTI and FTI IS because the MS/MS settings were matched.

As expected, the presence of a C-terminal stable isotope labeled K does not have a large impact on fragmentation behavior of FTI (IS).

By matching MS/MS settings for FTI and FTI IS, the latter can be used as internal standard as well as calibrant, see also the next 2 slides.

Ion ratiosFTI: 0.1-100 ng/mlFTI IS: 4 ng/ml

[M+2H]2+ => y7+ / y6

+ [M+2H]2+ => y7+ / a2

+ [M+2H]2+ => y6+ / a2

+

FTI FTI IS FTI FTI IS FTI FTI IS

Average (n=24 for FTI, n =24 for FTI IS)

1.72 1.72 2.68 2.69 1.56 1.56

RSD (%) 2.4 1.1 3.9 3.2 3.6 2.4

Results & Discussion (13)Comparison FTI and FTI IS absolute signals (1)

Response factor comparison. FTI 1 pg/ml to 100 ng/ml. FTI IS always 4 ng/ml

FTI: 27138 peak area per ng/ml (n=36, RSD 6.7%).

FTI IS: 28639 peak area per ng/ml (n=24, RSD 1.2%).

Experimental peak area ratio FTI IS / FTI = 1.06

Results & Discussion (14)Comparison FTI and FTI IS absolute signals (2)

Calculation theoretical peak area ratio (Bioanalysis 2016, 8, 891-904):(Fraction 1st isotope FTI IS / fraction 1st isotope FTI) multiplied by

(Molecular weight FTI / molecular weight FTI IS).(0.6289 / 0.5855) * (969.05 / 976.99) = 1.07

Theoretical peak area ratio = 1.07

Only 1.0 % bias compared to 1.06 experimental peak area ratio.

=> Absolute signals reproducible and similar between FTI and FTI IS.

Conclusions

Dynamic range at least 6 orders of magnitude.

Enhanced analyte base signal and higher precision at lower FTI concentrations through addition of FTI IS.

Only 1.00 fg or 1.03 amol FTI injected at lowest concentration 100 fg/ml.

Linear behavior between 1 and 100000 pg/ml.

Ion ratios and absolute signals reproducible and similar between FTI and FTI IS.