Establishment of the isotopically labeled LC-MS/MS method
A Shimadzu LCMS-8030 plus system, which exhibits high sensitivity and good reproducibility, was used for LC-MS/MS analyses. The initial attempt to select an appropriate high-performance liquid chromatography (HPLC) column focused on Waters Symmetry C18 and YMC-Pack ODS-AM, which are typical octadecylsilane (ODS) columns. A gradient system of water-acetonitrile (MeCN) was utilized for the elution solvent. As trifluoroacetic acid is less and might have some effects on the MS instruments, 0.1% (v/v) formic acid (FA) was used to enhance both column retention and ionization during MS. However, the reproducibility of the desmosine and isodesmosine peaks was not acceptable. Alternatively, we used a Supelco Discovery HS F5-3 pentafluorophenyl (F5)-type column, which exhibits unique selectivity characteristics due to p-p stacking interactions with aromatic compounds, delocalized electron density induced by fluorine, and weaker hydrogen bonds [33]. Using this column, the reproducibility of desmosine and isodesmosine peaks was improved using a water-acetonitrile gradient system with the addition of 0.1% (v/v) FA without an ion pairing reagent such as HFBA (Table S1). However, chromatographic separation of desmosine and isodesmosine was not accomplished under any LC conditions.
We then revised our strategy to optimize the tandem MS/MS method and establish appropriate equations for calculations. Major factors that can affect analytical sensitivity include selection of ions and the voltage of each MS compartment [34]. Optimizations were performed for general parameters, including the voltage of MS compartments with flow injection without a column. The program began with determination of the precursor ion followed by product ion search, which enumerates desirable product ions. The precursor ion was set according to the molecular weight of desmosine. A protonated double-charged ion (m/z 263.25) was selected as the precursor ion, and product ions exhibiting high intensity were selected in the second step. Voltage optimization was performed for these precursor and product ions automatically [35].
Based on observed m/z values and the structures of desmosine and isodesmosine, the structures of some fragment ions were estimated (Figure S1) using multi-reaction monitoring (MRM) mode. It should be noted that isodesmosine formed the same fragments, but the relative intensity of the ions enabled them to be distinguished from desmosine [36]. The m/z 232.10 and 397.25 ions were clearly different; the m/z 232.10 ion was easier to detect than the m/z 397.25 ion from desmosine. However, in the case of isodesmosine, the area of the m/z 397.25 ion peak was larger than that of the m/z 232.10 ion peak. Although the 84.15 m/z ion peak exhibited the greatest area among the peaks of both desmosine and isodesmosine, it was not a favorable ion because peaks with a low m/z value are often associated with noise resulting from impurities such as peptides or plasticizers. Therefore, the product ions m/z 232.10 and 397.25 exhibiting high intensity formed from precursor ion m/z 263.65 were determined to be the best targets for detecting desmosine and isodesmosine. The optimization was also applied to isodesmosine-13C3,15N1 (Figure S2). Detected ions at m/z 265.65 and 401.25 corresponded to m/z 263.65 and 397.25 peaks of isodesmosine. Optimized MS/MS conditions for desmosine, isodesmosine, and isodesmosine-13C3,15N1 are summarized in Table S2.
In order to analyze human plasma samples, calibration curves were drawn for fragment ions m/z 232.10 and 397.25 with isotopic internal standard isodesmosine-13C3,15N1. An example calibration sample (0.005 ppm) is shown in Figure S3. As shown in the MS chromatogram, two fragments (m/z 232.10 and 397.25) were clearly observed. In all samples, the retention times of the internal standard and isodesmosine exhibited good reproducibility at approximately 12 min. The peak area ratios between the isotopic standard and isodesmosine were calculated to draw calibration curves (Figures S4 and S5). In order to obtain greater accuracy, calibration curves were drawn for each sample group. Satisfactory Rr1 (correlation coefficient value) and Rr2 (coefficient of determination value) indicated that all calibration points were successfully analyzed. Compared with the curve for fragment ion m/z 232.10, fragment ion m/z 397.25 exhibited better accuracy in both stroke and control samples, as the function of fragment ion m/z 397.20 was closer to the zero point, and Rr2 was >0.999. Therefore, product ion m/z 397.25 was selected for the calibration of isodesmosine.
The reproducibility of calibration samples was confirmed using the m/z 397.25 ion (Tables 1 and S3). The mean concentration indicates the concentration of a sample as predicated from the calibration curve. Standard deviation, relative standard deviation, and signal-to-noise (S/N) ratio were also calculated. The limit of quantitation (LOQ) was determined at S/N = 10 unless otherwise stated in Table S3. According to the analytical results, the LOQ of isodesmosine based on the m/z 397.25 fragment was 0.005 ppm for stroke samples and 0.01 ppm for healthy control samples. This difference derived from instrument or column conditions, because the two calibration curves were drawn before the respective analyses, which were carried out on different days.
Table 1
Reproducibility of calibration samples for stroke (fragment: m/z 397.25).
Concentration of isodesmosine (ppm)
|
Area ratio
|
Mean concentration
(ppm)
|
Area ratio
SD
|
Area ratio
RSD
|
0.005
|
0.0631258
|
0.00917
|
0.0179
|
0.4218
|
|
0.0333533
|
0.00473
|
|
0.030962
|
0.00437
|
0.01
|
0.0705102
|
0.01027
|
0.0074
|
0.1094
|
|
0.0589324
|
0.00854
|
|
0.0726177
|
0.01059
|
0.02
|
0.139143
|
0.02052
|
0.0062
|
0.0456
|
|
0.128068
|
0.01886
|
|
0.138298
|
0.02039
|
0.05
|
0.31623
|
0.04695
|
0.0094
|
0.0290
|
|
0.32687
|
0.04853
|
|
0.335067
|
0.04976
|
0.1
|
0.696939
|
0.10377
|
0.0193
|
0.0285
|
|
0.675204
|
0.10052
|
|
0.658539
|
0.09804
|
SD: standard deviation; RSD: relative standard deviation. |
Measurement of the plasma concentrations of desmosine and isodesmosine in acute stroke patients using isotope-dilution LC-MS/MS
A total of 17 plasma samples obtained from stroke patients (samples S1-S9) and healthy control subjects (samples C1-C8) were hydrolyzed and purified as previously reported [11-13]. A comparison of the peak area ratios of desmosine and isodesmosine with the internal standard is shown in Table 2. Raw values for fragment ions m/z 232.10 and 397.25 are shown in the Supporting Information (Tables S4 and S5). Quantitative analysis of desmosine and isodesmosine was possible for all samples in which the peak area could be determined for both the m/z 232.10 and 397.25 channels. Therefore, peak areas for desmosine and isodesmosine were obtained for seven samples (S1-S3, S6, S8, S9, and C3). Four samples (S4, S5, S7, and C7) were detected in the m/z 397.25 channel, but they were below the LOQ due to lack of detection of the m/z 232.10 channel. Hence, the peak area ratio of desmosine and isodesmosine in these samples (S4, S5, S7, and C7) was above the limit of detection (LOD) but under the LOQ. In summary, the areas of desmosine and isodesmosine in all plasma samples from stroke patients were above the LOD. Quantitative analysis of desmosine and isodesmosine was thus possible for six of nine samples from stroke patients (S1, S2, S3, S6, S8 and S9). Among the eight healthy control group samples, the peak area ratio of desmosine and isodesmosine was above the LOD in two samples (C3 and C7). Quantitative analysis of desmosine and isodesmosine was only possible for one control sample (C3); the other five samples (C1, C2, C4, C5, and C8) were below the LOD.
Table 2
Peak area ratio of desmosine and isodesmosine compared with the internal standard.
|
Desmosine |
Isodesmosine |
Desmosine + Isodesmosine
|
Comment |
S1
|
0.03701812
|
0.031877437
|
0.068895558
|
|
S2
|
0.014672097
|
0.047289901
|
0.061961998
|
|
S3
|
0.024437845
|
0.03809611
|
0.062533955
|
|
S4
|
-
|
-
|
<LOQ
|
Fragment 232.15 was ND.
|
S5
|
-
|
-
|
<LOQ
|
Fragment 232.15 was ND.
|
S6
|
0.036334632
|
0.017706312
|
0.054040945
|
|
S7
|
-
|
-
|
<LOQ
|
Fragment 232.15 was ND.
|
S8
|
0.02580029
|
0.030987129
|
0.056787419
|
|
S9
|
0.02564125
|
0.02081456
|
0.04645581
|
|
C1
|
-
|
-
|
<LOD
|
Fragments 232.15/397.25 were ND.
|
C2
|
-
|
-
|
<LOD
|
Fragments 232.15/397.25 were ND.
|
C3
|
0.015242721
|
0.031290451
|
0.046533172
|
|
C4
|
-
|
-
|
<LOD
|
Fragments 232.15/397.25 were ND.
|
C5
|
-
|
-
|
<LOD
|
Fragments 232.15/397.25 were ND.
|
C6
|
-
|
-
|
<LOD
|
Fragments 232.15/397.25 were ND.
|
C7
|
-
|
-
|
<LOQ
|
Fragment 397.25 was ND.
|
C8
|
-
|
-
|
<LOD
|
Fragments 232.15/397.25 were ND.
|
*All values refer to mean area ratio obtained from calibration curves. S: stroke; C: control; LOQ: limit of quantitation; LOD: limit of detection; ND: not detected. |
Based on the peak area ratio, the concentration of desmosine, isodesmosine, and their total amount in plasma were calculated (Figures 2 & 3 and Table S6). The relationship between the amount of desmosine and the patient’s pathology was also confirmed. Plasma concentrations of desmosine and isodesmosine were elevated in stroke patients compared with healthy volunteers (0.05810 vs. 0.005817, unpaired t-test, P<0.05. Figure 3). In contrast, there were no obvious differences in desmosine concentration between ischemic versus hemorrhage stroke patients (representative cases are shown in Figure 4).
Several issues must be addressed to interpret desmosine and isodesmosine levels in plasma (Figures 1 and 2). First, the number of plasma samples analyzed was small. Second, elevated plasma elastin levels are not solely derived from vascular injuries caused by acute cerebral stroke. A history of COPD and smoking habit can affect elastin dynamics. Plasma concentrations of desmosine and isodesmosine should therefore be carefully interpreted in patients who have a history of COPD or smoking habit. Finally, the optimal time point for measuring plasma elastin levels remains unclear. The progression of a brain injury depends on a variety of factors, such as stroke subtype and location of the stroke. Future studies including larger cohorts should be conducted to validate the findings of the present study.
Based on these results, we conclude that the plasma of stroke patients contains increased levels of desmosine and isodesmosine due to vascular injuries. The present research suggests that desmosine and isodesmosine could be used as novel biomarkers for vascular injuries after acute cerebral stroke. Further studies should be conducted to validate the diagnostic value of desmosine and isodesmosine measurements for evaluating vascular injuries caused by acute cerebral stroke.