Practical comparison of LC-MS/MS-based workflows for quantitation of desmosine and isodesmosine in urine – potential lung damage biomarkers in premature infants Karolina M. Krasinskaa, Carlos Millab, Stephen R. Lynchc , Allis S. Chiena aVincent Coates Foundation Mass Spectrometry Laboratory; bDepartment of Pediatrics; cNMR Facility; Stanford University, Stanford, CA
Overview LC-MS/MS platforms Results TABLE 1. Comparison of technical features. Analysis of DES and IDES in straight urine is only possible with methods A and C. This work compares the feasibility of three LC-MS/MS- Instrumentation FIGURE 2. Comparison of LC conditions. Three mobile phase Strong matrix effect and poor sensitivity has been observed when modifiers were evaluated for DES/IDES separation in matrix free using method B. Of all three, method A utilizes the most mass based workflows for the quantitation of desmosine (DES) LC system: Agilent HP 1100 samples: 0.1% formic acid (A), 5 mM HFBA/5 mM AA (B) and spectrometry compatible LC mobile phases but does not allow and isodesmosine (IDES) in urine samples MS detection: Waters Quattro Premier triple quadrupole MS 0.5% TFA (C). With each eluent system, several gradient, baseline resolution of DES and IDES. Ionization mode: positive ESI Each of the tested methods, with its advantages and temperature and flow rate settings were tested. The best results Scanning mode: Selected Reaction Monitoring (SRM) disadvantages, proves to be suitable for quantitative for each modifier and its respective column are presented below. Comparison criterion ABC
analysis *) LC conditions 1uM DES/IDES, 0.1uM PYR IS new Hypercarb, 100x2.1mm, 3um, A: 0.1% FA in water, B: 0.1% FA in MeOH LLOQ in solvent / urine 5 / 10 fmol 5-10 / 200 fmol 50 / 5 fmol MillaC_130118_40328_15 Sm (Mn, 1x1) MRM of 6 Channels ES+ 4.60 526.3 > 83.6 (DES/IDES) 5.42e5 Time – Sample analysis 10 min 10 min 10 min Time IDES AB C %B 4.71 [min] DES Chromatographic resolution Not sufficient for Introduction baseline baseline 02 Hypercarb Atlantis T3 Hypercarb (DES/IDES separation quantitation 0.3 2 Column (Thermo Fisher Scientific) (Waters) (Thermo Fisher Scientific) Hypercarb Matrix interference **)
% Minor Significant Not observed Desmosine (DES) and isodesmosine (IDES) are lysine- 100 mm x 2.1 mm, 3 µm 2.1 mm x 100 mm, 3 µm 100 mm x 2.1 mm, 3 µm A 640 (urine – no sample prep) 740 0.1% FA derived pyridinium amino acids, byproducts of the Column Hypercarb Atlantis T3 Hypercarb Mobile Phase A 0.1% FA 5 mM HFBA/ 5 mM AA 0.5% TFA 7.5 2 destruction of elastin. Release of these two compounds to 10 2 Mobile phase modifier 0.1% FA 5 mM HFBA/5 mM AA 0.5% TFA 5 mM HFBA/ 5mm AA in 0 Time Mobile Phase B 0.1% FA in acetonitrile 0.5% TFA 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 body fluids serves as an indicator of elastin degradation 80% acetonitrile Flow rate [µl/min] 250 250 300 100nM DES/IDES, Atlantis T3, 100x2.1mm, 3um, A: 5mM HFBA/5mM AA, B: 5mM HFBA/5mM AA/80% Acetonitrile associated with lung damage in bronchopulmonary MillaC_130116_40328_77 Sm (Mn, 3x2) MRM of 6 Channels ES+ *) matrix interferences made sensitivity assessment inaccurate and irreproducible Flow rate 250 µL/min 250 µL/min 300 µL/min 5.52 526.3 > 83.6 (DES/IDES) 6.32e4 **) method B is not feasible for analysis of straight urine. In order to achieve good sensitivity and reproducibility dysplasia (BPD), a significant cause for morbidity and Time IDES 1 %B biological sample need extensive sample preparation (solid phase extraction) as described by Ma et al. mortality among infants born prematurely. This study FA: formic acid; HFBA: heptafluorobutyric acid; AA: ammonium acetate; TFA: trifluoroacetic acid [min] 01 DES 5.96 evaluates three LC-MS/MS based workflows – differing FIGURE 1. Analytes. Desmosine (DES) and Isodesmosine 0.3 1 Atlantis T3 mainly in chromatography – for quantitation of DES and (IDES) are positional isomers which generate nearly identical CID B % 620 Conclusions 7.5 20 5 mM HFBA/AA IDES. Comparison criteria include sensitivity, fragmentation patterns. Due to this similarity, MS analysis is not 81 Each of the tested methods can be applied to the quantitative chromatographic resolution, matrix interference, sufficient to distinguish between these two isomers. The H-NMR 10 1 analysis of DES and IDES in urine samples – however, each has 0 Time robustness, and total speed of analysis. Key goals were spectra of DES and IDES reveal significant differences for the 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 advantages and disadvantages which must be considered in pyridine ring protons at high field [ppm]. 500nM DES/IDES, 500nM PYR IS, 0.1%FA context of project requirements: reduction of sample preparation time and use of volatile LC MillaC_130521_42199_36 Sm (Mn, 3x2) MRM of 6 Channels ES+ 7.62 526.3 > 83.6 (DES/IDES) 2.83e4 Methods B and C achieve baseline resolution of two isomeric Time IDES eluents. This study illustrates practical choices which must %B [min] DES analytes. However, current clinical data do not support the be made in developing and selecting a fit-for-purpose 7.97 DES IDES 010 necessity of quantifying DES and IDES individually – so analytical method. 0.3 10 Hypercarb C % 630 chromatographic separation, while satisfying analytically, may 4 730 0.5% TFA be extraneous. 7.5 10 8.18 Method A is the most MS compatible and would be best suited 10 10 Sample preparation 6 2 6 1 Time for large scale measurement of combined DES and IDES, as its 3.00 4.00 5.00 6.00 7.00 8.00 9.00 run time can be significantly shortened to provide high- Standards: throughput analysis. Powdered desmosine (DES) and FIGURE 3. Analysis of human urine samples. Urine samples Both methods B and C utilize ion paring agents; of the two, TFA isodesmosine (IDES) were purchased from 33 infants were analyzed using method A. The cohort is more MS friendly than HFBA, in that it is more volatile, easier from Elastin Product Company. includes 18 premature and 15 full term newborns. Of the to wash out, and causes less ionization suppression. IDES_NEW_MSMS_02 19 (0.452) Cm (2:20) Daughters of 526ES+ DES_SIGMA_MSMS_02A 12 (0.322) Sm (Mn, 2x1.00); Cm (3:29) Daughters of 526ES+ 397.2 2.63e7 481.3 1.61e6 Successful application of Method B requires extensive, time Acetylated pyridinoline was obtained from 100 526.4 100 83.7 premature babies, 7 developed bronchopulmonary dysplasia Quidel as a 3.8 µM solution (in 90% (BPD) and 11 did not. No obvious correlation among disease consuming sample preparation, while methods A and C work 526.3 397.2 state, age and prematurity was observed in this set of samples. well with straight urine. % % 352.2 acetic acid) and used as an Internal 352.1 527.3 83.6 436.3 PYR IS 481.1 129.8 Due to its sensitivity, chromatographic resolution and lack of 129.9 Standard (PYR IS). 85.7 392.0 291.0 308.2 191.9 247.1 307.4 matrix interferences, Method C is our choice for this ongoing 0 m/z 0 m/z Stock solution: 1 mM in 50% acetonitrile/0.1% formic acid 50 100 150 200 250 300 350 400 450 500 550 600 100 150 200 250 300 350 400 450 500 550 600 project.
Working solutions: 50 µM; prepared by dilution of stock Analyte Abbreviation MW SRM LC-MS/MS solution with water Calibration curve: 0.1 nM – 5000 nM prepared by serial Desmosine DES 525.3 526.3 > 83.6, 397.1, 481.3 Future work dilutions in matrix-free solvent or urine Isodesmosine IDES 525.3 526.3 > 83.6, 397.1, 481.3 The measured levels of DES/IDES in infant urine did not Biological samples: Urine samples were collected from Acetylated pyridinoline PYR IS 470.1 471.1 > 81.8, 128.0, 267.2 support the use of DES/IDES as biomarkers for BPD. Future 33 human infants: 15 normal controls and 18 premature work will concentrate on the analysis of plasma and airway fluid H-C6 samples using the established LC-MS/MS workflow. babies. Of the premature babies, 7 developed lung disease H-C2
and 11 did not. Creatinine levels were used for H-C6 References H-C4 normalization purposes. 1) Ma,S.; Turino,G.; Lin,Y.Y. J.Chrom.B 2011, 879: 1893-1898 Acknowledgements Thanks to the Vincent and Stella Coates Foundation and NIH Grant Award Number 1P01HL108797. All biological samples were injected as-is, without any Thanks to Anne Coates, MD and Julie Kim, MD for sample procurement. sample cleanup or dilution. This poster may be downloaded from the Stanford University Mass Spectrometry website at http://mass-spec.stanford.edu/Publications.html
61st ASMS Conference, June 9-13, 2013, Minneapolis, MN