Comparison of Full Scan MS2 and MS3 Linear Ion Trap Approaches for Quantitation of Vitamin D

Comparison of Full Scan MS2 and MS3 Linear Ion Trap Approaches for Quantitation of Vitamin D Julie A. Horner 1, Marta Kozak 1, Subodh Nimkar 1, and August A. Specht 1 1 Thermo Fisher Scientific, San Jose, CA

Overview Purpose: Comparison of ion trap full scan methods for quantitation of 25-OH Vitamin D2 and D3 in human plasma samples. Methods: Liquid-liquid extraction followed by LC-MS2 or LC-MS3 analysis of fresh plasma samples. Results: Both methods are acceptable for Vitamin D quantitation. Full scan MS2 provides superior sensitivity whereas full scan MS3 provides superior selectivity. Introduction There has been a growing interest in Vitamin D and its role in human metabolism. Vitamin D is metabolized to 25-OH D in the liver. Total vitamin D is best determined by measuring total 25-hydroxyvitamin D (D 2 and D 3 ) in serum because the half-life of 25- OH D is about three weeks with serum concentrations of 1 5. Vitamin D supplementation in both food and tablets comes in both the D 2 and D 3 forms, making it imperative to measure 25-OH D 2 and 25-OH D 3. Although optimal serum concentrations of total 25-OH D are generally agreed to be 3, there is considerable discussion on the serum concentration of 25-OH D considered to be inadequate for bone and overall health, but < is generally regarded as deficient. Serum concentrations >1 are generally regarded as potentially toxic. We have developed dual pressure linear ion trap methods based on Full Scan MS2 and Full Scan MS3 for maximum sensitivity and selectivity and therefore precision and accuracy in measurement of 25-OH and D3. Methods Sample Preparation Plasma samples were prepared at a university clinical laboratory using the liquid-liquid extraction procedure described below: 1. 25 ul each sample is treated with ul of.1 M NaOH, vortexed and left at room temperature for 15 minutes. 2. 1 ul of internal standard is added and vortexed 3. 2 ml of n-heptane is added and vortexed 1 minute. 4. The mixture is centrifuged at 15 rpm for 4 minutes. 5. The tubes are placed in a dry ice:acetone bath until the aqueous layer freezes; the top layer is then discarded. 6. The remainder is dried under nitrogen at 37 C and reconstituted with 1 ul of : mobile phase. Liquid Chromatography Liquid-liquid extracted plasma samples were injected using a Thermo Scientific Accela Open AS and separated using a reversed-phase gradient delivered by a Thermo Scientific Accela 125 uhplc pump. The 5 minute method uses a 2.1x5 mm Phenomenex Luna c18 column and a flow rate of ul/min. Both gradient and column were duplicated from a clinical laboratory. Mass Spectrometry Atmospheric pressure chemical ionization source conditions were optimized for best signal to noise. Mass spectrometric detection on a dual pressure linear ion trap was achieved using 3 Full Scan MS3 or MS2 scan events for the water loss of 25-OH, 25-OH and the internal standard 25- OH -d6.. Data Analysis All data were processed using Thermo Scientific LC Quan 2.6.1 Quantitation software. 33.7 in plasma 1.5 in plasma 2 Comparison of Full Scan MS2 and MS3 Linear Ion Trap Approaches for Quantitation of Vitamin D

Results Challenges in Vitamin D Analysis Vitamin D is typically not found free in serum samples due to a high probability of protein binding and thus poses a challenge for sensitive and reproducible high performance liquid chromatography (HPLC). Additionally, various serum sample matrix constituents are found to cause ion suppression that reduces accuracy and reproducibility during real sample analyses. This ion suppression can be especially troublesome with rapid LC mass spectrometry (MS) methods where insufficient resolution can be observed between matrix contaminants and analyte peaks. 25-OH 25-OH C:\j.horner\...\Raw files\p-46 8/4/11 9:51:3 Relative Abundance 1 8 1 8 1 8 211.14 217. 229.17 245.2 243.19 211.15 229.15 239.18 213.19 225.17 9.12 211.14 227.14 229.17 21 2 23 2 m/z Vitamin D Quantitation Using Full Scan MS2 In addition, Vitamins D2 and D3 are fairly linear structures resulting in multiple fragmentation channels and distributed ion current in MS2 experiments. The full scan MS2 total and base peak extracted ion currents for 25-OH, 25-OH Vitamin D3 and the internal standard shown in Figures 1 a and b respectively show an improvement in S/N with approximately five-fold difference in intensity. FIGURE 1a. Full Scan MS2 Total Ion Current Chromatograms for 25-OH (top), 25-OH - d6 (top) and 25-OH (bottom) for Sample 46. 1 8 1 8 1 8 1 8 2. 2.33 2.41 2.32 2. 33.7 2.33in plasma 2.65 1.5 in 2.5 plasma 1.57 2.29 2.4 1.5 2. 2.5 3. 3.5 NL: 1.57E7 TIC NL: 1.57E7 Full MS2 383.5@cid29. [.-28.] NL: 2.96E6 Full MS2 389.5@cid29. [.-28.] NL: 1.35E5 Full MS2 395.5@cid29. [.-28.] FIGURE 1b. Full Scan MS2 Base Peak Extracted Ion Chromatograms for 25-OH (top), 25-OH -d6 (middle) and 25-OH (bottom) for Sample 46. 1 8 1 8 1 8 1 8 2.33 2.32 2. 2.41 2. RT: SN: 5114 2.61 RT: SN: 8813RMS RT: 2.65 SN: 398 1.5 2. 2.5 3. 3.5 NL: 1.57E7 TIC NL: 3.88E5 m/z= 256.72-257.72 Full MS2 383.5@cid29. [.-28.] NL: 4.22E5 m/z= 262.72-263.72 Full MS2 389.5@cid29. [.-28.] NL: 2.E4 m/z= 268.72-269.72 Full MS2 395.5@cid29. [.-28.] However, the increase in selectivity of the base peak extracted in chromatograms is necessary for accurate peak identification and quantitation of target levels. Using the total ion current of the full scan MS2 experiment, even with a restricted product ion mass scan range, results in significant interference. 1 8 1 8 1 8 1 8 FIGURE 3. Standard Curves(right) for obtained using Full Scan MS2. Accura tables (left). 3.3-3.31 7.5 6.51 14.1 4.43 29.7-5.57 51.2-2.6 2.6-4.4 5.3 1.18 12. -5.43 25.5 5.9 5.3-5.81 1.4 1.2 1..8.6.4.2. The peak top full scan MS2 mass spectra shown in Figure 2 demonstrate the issue surrounding multiple fragmentation channels, particularly for 25-OH and the internal standard. These additional channels result in noise in the chromatogram as well as reduced sensitivity for the extracted ion chromatograms. Vitamin D Quantitation Using Full Sca The majority of LC-MS methods for Vitam types. The sensitivity and simplicity of a desirable for any analysis; however, as s chromatograms, selectivity is also require there can be interference in a given trans such as plasma. Thermo Scientific Poster Note PN63565_E 6/12S 3

FIGURE 2. Peak Top Full Scan MS2 Spectra for 25-OH (top), Internal Standard (middle), and 25-OH (bottom) for Sample 46. C:\j.horner\...\Raw files\p-46 Relative Abundance 8/4/11 9:51:33 PM 257.21 NL: 2.37E5 1 P-46#796-813 RT: 2.58-2.61 8 383.5@cid29. [.-28.] 271.22 229.17 245.22 211.14 243.19 269.22 273.23 217. 263.24 NL: 2.91E5 1 P-46#796-81 RT: 2.58-2.61 AV: 5 F: ITMS + c APCI 8 389.5@cid29. [.-28.] 253. 229.15 211.15 271. 239.18 257.17 277.24 213.19 225.17 269.19 NL: 1.64E4 1 P-46#816-834 RT: 2.63-2.66 8 227.14 395.5@cid29. [.-28.] 9.12 271. 251.18 265.22 211.14 229.17 257. 279.18 21 2 23 2 25 2 27 28 m/z The Vitamin D analysis is done for a set of real samples. There is enough material for each quality control (4 total), standard (5 total) and real sample (9 total) to be injected just once. Our clinical laboratory collaborators carry out the analysis and send the same sample set to us for comparison. Figure 3 shows the standard curve linearity and accuracy for both 25-OH and 25-OH done using our full scan MS2 method. The analysis of Vitamin D in fresh human plasma samples was successfully carried out using ion trap based full scan MS2; both accuracy and limits of quantification are within desired specifications. FIGURE 3. Standard Curves(right) for 25-OH and 25-OH obtained using Full Scan MS2. Accuracies for each Standard are listed in the tables (left). 3.3-3.31 7.5 6.51 14.1 4.43 29.7-5.57 51.2-2.6 1.4 1.2 1..8.6.4.2 Y =.2+.2*X R^2 =.9953 W: 1/X^2. 5 1 15 25 3 35 45 5 55 FIGURE 4a. Full Scan MS3 Total Ion Current Chromatograms for 25-OH, 25-OH and 25-OH -d6 f or Sample 45. 1 2.58 8 1 8 1 8 1 8 2.57 2.56 2.49 23.6 in plasma 2.58 d6 2.63 1. in plasma 1.5 2. 2.5 3. 3.5 TIC MS p-45_82211 Full MS3 383.5@cid29. 257.@cid24. [1.-28 p-45_82211 Full MS3 389.5@cid29. 263.@cid26. [1.-28 p-45_82211 NL: 1.38E4 Full MS3 395.5@cid29. 269.@cid26. [1.-28 p-45_82211 In this study, we use a full scan MS3 ap most intense product ion from the MS2 experiment which is inherently selective also sensitive. The total ion current chromatograms an experiments are shown in Figures 4a an the MS3 experiment is sufficiently selec level of detection, 1 (bottom trace the base peak extracted ion chromatogr noise ratios for all components are with scan MS2 experiment. The combination of sensitivity and selec and D3 (Figure 5) and accu scan MS2 experiment (Figure 2). 1.4 1.2 Y =.217+.274*X R^2 =.9911 W: 1/X^2 chromatograms is et levels. Using the ricted product ion 2.6-4.4 5.3 1.18 12. -5.43 25.5 5.9 5.3-5.81 1..8.6.4.2. 5 1 15 25 3 35 45 5 55 nstrate the issue H and the well as reduced Vitamin D Quantitation Using Full Scan MS3 The majority of LC-MS methods for Vitamin D are based on MS2 (including SRM) scan types. The sensitivity and simplicity of a total ion current measurement is highly desirable for any analysis; however, as seen in the TIC versus base peak extracted ion chromatograms, selectivity is also required. Even when the SRM experiment is used, there can be interference in a given transition particularly in a complex sample matrix such as plasma. 4 Comparison of Full Scan MS2 and MS3 Linear Ion Trap Approaches for Quantitation of Vitamin D

NL: 2.37E5 P-46#796-813 RT: 2.58-2.61 383.5@cid29. [.-28.] NL: 2.91E5 P-46#796-81 RT: 2.58-2.61 AV: 5 F: ITMS + c APCI 389.5@cid29. [.-28.] NL: 1.64E4 P-46#816-834 RT: 2.63-2.66 395.5@cid29. [.-28.] FIGURE 4a. Full Scan MS3 Total Ion Current Chromatograms for 25-OH, 25-OH and 25-OH -d6 f or Sample 45. 1 2.58 8 1 8 1 8 1 8 2.57 2.56 2.49 23.6 in plasma 2.58 d6 2.63 1. in plasma 1.5 2. 2.5 3. 3.5 TIC MS p-45_82211 Full MS3 383.5@cid29. 257.@cid24. [1.-28.] p-45_82211 Full MS3 389.5@cid29. 263.@cid26. [1.-28.] p-45_82211 NL: 1.38E4 Full MS3 395.5@cid29. 269.@cid26. [1.-28.] p-45_82211 FIGURE 4b. Full Scan MS3 Total Ion Chromatograms for 25-OH vitamin D2, 25-OH and Full Scan MS3 Base Peak Extracted Ion Chromatogram for 25-OH -d6 for Sample 45. 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 2.57 2.56 2.58 RT: SN: 1123RMS RT: 2.58 SN: 3319RMS RT: 2.63 SN: INF 1.5 2. 2.5 3. TIC MS p-45_82211 Full MS3 383.5@cid29. 257.@cid24. [1.-28.] p-45_82211 Full MS3 389.5@cid29. 263.@cid26. [1.-28.] p-45_82211 NL: 6.59E3 m/z= 25.74-251.74 Full MS3 395.5@cid29. 269.@cid26. [1.-28.] p-45_82211 3.5 Vitamin D Anaylsis in Real Samples. We analyzed several sets of samples a shown) and MS3 (Figure 6). Good corr and (bottom). The exceptio horizontal axis were reported as below real values by Thermo. 5 3 1 1 In this study, we use a full scan MS3 approach for data acquisition. We chose the most intense product ion from the MS2 spectrum for each target to give an MSn experiment which is inherently selective and, through reduction of noise contributions, also sensitive. 5 5-OH are listed in the The total ion current chromatograms and signal to noise ratios for the full scan MS3 experiments are shown in Figures 4a and 4b. Immediately of note is that the TIC in the MS3 experiment is sufficiently selective to use for quantitation, even at the lowest level of detection, 1 (bottom trace Figure 4a) although for this study we use the base peak extracted ion chromatogram (bottom trace Figure 4b). Also, signall to noise ratios for all components are within a factor of two of those obtained in the full scan MS2 experiment. The combination of sensitivity and selectivity gives quantification results for 25-OH and D3 (Figure 5) and accuracies comparable to those seen in the full scan MS2 experiment (Figure 2). 3 1 1 FIGURE 5. Standard Curves (right) for 25-OH and 25-OH obtained using Full Scan MS3. Accuracies for each Standard Level are Listed in the Tables (left). 4. Y =.98+.818*X R^2 =.9988 W: 1/X^2 =.9911 W: 1/X^2 3.3 1.54 7.5-3.33 14.1 -.24 3. 2. 29.7-1.47 1. 51.2 3.5. 5 1 15 25 3 35 45 5 55 35 45 5 55 including SRM) scan ment is highly e peak extracted ion experiment is used, mplex sample matrix 2.6-3.1 5.3 6.49 12. -.93 25.5 4.98 5.3-7.43 2. 1.5 1..5 Y =.42+.443*X R^2 =.9944 W: 1/X^2. 5 1 15 25 3 35 45 5 55 Thermo Scientific Poster Note PN63565_E 6/12S 5

3. TIC MS p-45_82211 Full MS3 383.5@cid29. 257.@cid24. [1.-28.] p-45_82211 Full MS3 389.5@cid29. 263.@cid26. [1.-28.] p-45_82211 NL: 6.59E3 m/z= 25.74-251.74 Full MS3 395.5@cid29. 269.@cid26. [1.-28.] p-45_82211 3.5. We chose the o give an MSn noise contributions, the full scan MS3 is that the TIC in, even at the lowest this study we use b). Also, signall to btained in the full esults for 25-OH seen in the full FIGURE 6. Correlation Plot of Full Scan MS3 Data Obtained on Velos Pro versus SRM Data Obtained at the Collaborator Site. Vitamin D Anaylsis in Real Samples. We analyzed several sets of samples against collaborator data using both MS2 (not shown) and MS3 (Figure 6). Good correlation is observed for both (top) and (bottom). The exceptions are several points at the origin of the horizontal axis were reported as below limit of quantification by the collaborator but as real values by Thermo. 5 3 1 1 3 5 Collaborator SRM Results 5 3 1 1 3 5 Conclusion Collaborator SRM Results Vitamin D2 Vitamin D3 The full scan MSn analysis of Vitamin D in plasma results indicate the following: Both full scan MS2 and full scan MS3 provide quantitation and accuracies suitable for analysis of Vitamin D in samples.. Full Scan MS2 provides higher sensitivity for this analysis, full scan MS3 provides better selectivity. Ion trap based methods can be used for quantitation of a small number of compounds in a complex matrix. These methods will be used to determine suitability for quantitation in singly crashed plasma rather than LLE plasma samples. Acknowledgements We would like to thank Kara Lynch and Shannon Kastner from the University of California - San Francisco for supplying fresh plasma sample sets. These methods are For Research use Only. Not for use in Diagnostic Procedures. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Comparison of Full Scan MS2 and MS3 Linear Ion Trap Approaches for Quantitation of Vitamin D

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