Analyte profiling by stochastic dynamics mass spectrometry for sewage sludge valorization (supporting information)

SUPPORTING INFORMATION TITLE: Analyte profiling by stochastic dynamics mass spectrometry for sewage sludge valorization Author: Bojidarka Ivanova 1 Figure S1. Chromatographic (A) and mass spectrometric (B) data on diclofenac (1) in biota according to [67]. 2 Figure S2. Chromatographic (A) and mass spectrometric (B) data on diclofenac (1) and citalopram (2) in biota blank sample according to [67]. 3 Figure S3. Chromatographic (A) and mass spectrometric (B) data on diclofenac (1) and citalopram (2) in sludge blank sample according to this work. 4 Figure S4. Chromatographic and mass spectrometric data on diclofenac (1) in QuEChERS sludge sample according to this work. 5 Figure S5. Mass spectrometric data on diclofenac (1) in sludge sample according to this work and accounting for between-day variability; chemical diagrams of species at m/z 295 and 296 depending on their proton accepting capability and tautomerism. 6 Figure S6. Functional relationships between D’’SD data on equation (2), respectively total intensity data on entire measurement time [arb.units] with respect to analyte concentrations [ng.ml-1]of diclofenac (1) and citalopram (2) using data on ions at m/z 296 and 325; chemometrics. Figure S7. Chromatographic data and CID-MS/MS spectra of diclofenac (1) at m/z 296 of calibration samples S0 and S1. 7 Figure S8. Chromatographic data and CID-MS/MS spectra of diclofenac (1) at m/z 296 of calibration samples S2 and S5. Figure S9. Linear relationships between average intensity data on entire measurement time or MPA parameters of ions at m/z 295 or 325 of CID-MS/MS spectra of diclofenac (1) or citalopram (2) in sludge with respect to concentrations of analytes; chemometrics. 8 Figure S10. CID-MS/MS spectra of citalopram (2) at m/z 325 of calibration samples S0, S1 and S2 per scan and over the entire measurement time. Figure S11. CID-MS/MS spectra of citalopram (2) at m/z 325 of calibration samples S3, S6 and S7. 9 Table S2 Experimental dataset of measurable variables of calibration samples S0, S1, and S5 of CIDMS/MS spectra of diclofenac (1) at m/z 296 Cali_S5_1.RAW Cali_S1_1.RAW Cali_S0_1.RAW Scan #: 2657 Scan #: 2652 Scan #: 2652 RT: 20.06 [mins] RT: 20.04 RT: 20.04 m/z I [arb.units] m/z I [arb.units] m/z I [arb.units] 294.8182 0 294.8182 0 294.8182 0 294.8847 0 294.8847 1407 294.8847 358 294.9513 0 294.9513 4010 294.9513 3227 295.0178 0 295.0178 3861 295.0178 7828 295.0843 0 295.0843 143 295.0843 7680 295.1508 142 295.1508 0 295.1508 381 295.2174 1485 295.2174 0 295.2174 40 295.2839 266 295.2839 0 295.2839 0 295.3504 0 295.3504 0 295.3504 0 295.417 0 295.417 0 295.417 0 295.4835 0 295.4835 0 295.4835 0 295.55 4 295.55 0 295.55 0 295.6165 0 295.6165 0 295.6165 0 295.6831 0 295.6831 0 295.6831 0 295.7496 0 295.7496 0 295.7496 0 295.8161 0 295.8161 0 295.8161 0 295.8827 0 295.8827 0 295.8827 0 295.9492 0 295.9492 0 295.9492 0 296.0157 0 296.0157 0 296.0157 0 296.0822 0 296.0822 0 296.0822 0 296.1488 0 296.1488 0 296.1488 0 16 Table S3 Experimental dataset of measurable variables of calibration samples S1, S6, and S7 of CIDMS/MS spectra of citalopram (2) at m/z 325 Cali_S7_1.RAW Cali_S6_1.RAW Cali_S1_1.RAW Scan #: 2025 Scan #: 2025 Scan #: 2025 RT: 15.05 [mins] RT: 15.05 RT: 15.05 m/z I [arb.units] m/z I [arb.units] m/z I [arb.units] 324.4 0 324.4 0 324.4 0 324.4667 0 324.4667 0 324.4667 0 324.5333 0 324.5333 0 324.5333 346 324.6 0 324.6 0 324.6 2959 324.6667 0 324.6667 98 324.6667 5659 324.7333 0 324.7333 1229 324.7333 3932 324.8 56 324.8 4502 324.8 0 324.8667 1085 324.8667 3264 324.8667 0 324.9333 3914 324.9333 587 324.9333 0 325 1784 325 59 325 0 325.0667 284 325.0667 0 325.0667 2 325.1333 54 325.1333 0 325.1333 0 325.2 0 325.2 0 325.2 0 Table S4 Experimental dataset of measurable variables of calibration samples S2, S6, and S7 of CIDMS/MS spectra of citalopram (2) at m/z 325; parameters of equation (2); descriptive statistics Cali_S7_1.RAW Cali_S7_1.RAW Cali_S7_1.RAW Scan #: 2165 Scan #: 2045 Scan #: 2025 RT: 16.17 RT: 15.22 RT: 15.05 Scan m/z I [arb.units] m/z I m/z I m/z I 2165 324.9667 569.375 324.7333 0 324.7333 431 324.7333 0 2045 324.9667 1520.25 324.8 0 324.8 1902 324.8 56 2025 324.9667 897.125 324.8667 0 324.8667 4330 324.8667 1085 mean 324.9667 - 324.9333 433 324.9333 5083 324.9333 3914 Sd(yEr±) 0 - 325 2313 325 416 325 1784 Se(yEr±) 0 - 325.0667 1373 325.0667 0 325.0667 284 <I> - 995.5833 325.1333 377 325.1333 0 325.1333 54 <I>2991186.2 325.2 59 325.2 0 325.2 0 <I2> - 1.15.106 <I2>–<I>2155549.8 D’’SD - 4.10.10-12 Cali_S2_1.RAW Cali_S2_1.RAW Cali_S2_1.RAW Scan #: 2115 Scan #: 2105 Scan #: 2055 RT: 15.76 RT: 15.68 RT: 15.29 17 Scan m/z I [arb.units] m/z I m/z I m/z I 2115 324.9 1140,625 324.6667 0 324.6667 955 324.666 7 382 2105 324.9 804 3.25.1020 324.7333 2417 324.733 3 2375 2055 324.9 839 324.8 196 324.8 2040 324.8 2911 mean 324.9 - 3.25.1021034 324.8667 806 324.866 7 1040 Sd(yEr±) 0 - 324.9333 3316 324.9333 214 324.933 3 0 Se(yEr±) 0 - 325 3243 325 0 325 0 <I> - 927.875 325.0667 1072 325.0667 0 325.066 7 1 <I>2860952 325.1333 264 325.1333 0 325.133 3 3 <I2> - 883787.5 <I2>–<I>222835.45 D’’SD - 6.00.10-13 Cali_S6_1.RAW Cali_S6_1.RAW Cali_S6_1.RAW Scan #: 2115 Scan #: 2095 Scan #: 2025 RT: 15.77 RT: 15.61 RT: 15.05 Scan m/z I [arb.units] m/z I m/z I m/z I 2115 325 1079 324.6667 0 324.666 7 321 324.666 7 98 2095 325 1541 324.7333 0 324.733 3 3024 324.733 3 1229 2025 325 885.3636 324.8 0 324.8 7007 324.8 4502 mean 325 - 324.8667 171 324.866 7 6272 324.866 7 3264 Sd(yEr±) 0 - 324.9333 1325 324.933 3 200 324.933 3 587 Se(yEr±) 0 - 325 2656 325 108 325 59 <I> - 1168.455 325.0667 838 325.066 7 19 325.066 7 0 <I>21.37.106325.1333 1154 325.133 3 0 325.133 3 0 <I2> - 1.44.106325.2 2450 325.2 0 325.2 0 <I2>–<I>275649.96 325.2667 2302 325.266 7 0 325.266 7 0 D’’SD - 2.00.10-12 325.3333 973 325.333 3 0 325.333 3 0 Table S5 Experimental dataset of measurable variables of calibration samples S2, S6, and S7 of CIDMS/MS spectra of citalopram (2) at m/z 325 over RT=15.04–19.99 mins 18 Cali_S2_1.RAW Cali_S6_1.RAW Cali_S7_1.RAW Scans: 2025–2645 Scans: 2025–2645 Scan s: 2025–2645 RT: 15.04–19.98 RT: 15.05–19.98 RT: 15.05–19.99 m/z I [arb.units] r.i. [%] m/z I [arb.units] r.i. [%] m/z I [arb.units] r.i. [%] 318.6 7 75.6 32.85 278.67 156.9 36.64 187.73 141.4 36 319 75.5 32.79 278.73 232.5 54.3 187.8 142.1 36.17 319.4 7 97.5 42.38 278.8 221 51.62 225.73 161.3 41.07 319.5 3 106.4 46.23 295.27 157.8 36.84 296.67 208.6 53.09 320.0 7 92.5 40.18 296.87 186.3 43.5 296.73 144.9 36.89 320.1 3 94.5 41.05 301.27 192 44.83 306.2 238.2 60.64 324.1 3 92 39.98 301.33 195.1 45.56 306.27 143 36.4 324.2 7 60.7 26.37 304.6 193.7 45.22 306.8 209.6 53.36 324.3 3 136.9 59.47 315.47 180.1 42.06 306.87 161.8 41.18 324.4 111.1 48.29 324.53 169.4 39.55 324.53 289.6 73.73 324.5 3 100.8 43.79 324.67 198.2 46.29 324.6 392.8 100 324.6 116.1 50.46 324.73 292.7 68.34 324.67 298.3 75.94 324.6 7 93.5 40.61 324.8 428.2 100 324.73 169.4 43.13 324.7 3 150.7 65.48 324.87 384.2 89.71 324.8 152.9 38.93 324.8 172.8 75.09 324.93 276.7 64.62 324.87 233 59.33 324.8 7 198.7 86.36 325 409 95.51 324.93 300.4 76.49 324.9 3 230.1 100 325.07 300.2 70.11 325 190.2 48.42 325 212.1 92.17 325.13 162 37.82 341.6 141.8 36.11 325.0 7 208.6 90.63 334.73 177.5 41.46 342.53 177.3 45.12 325.1 3 184.2 80.05 342.93 187.6 43.8 342.6 212.2 54.01 Table S6 Frequency analysis (M062X/SDD) of species of analytes using the Z-matrixes in the preceding table in GS and TSs used to compute parameters of equation (3) 19 Cit_307_a GS TS Freq. 24.7508 33.6239 48.4762 -106.5765 25.8467 37.5764 Freq. 65.3460 76.6889 91.8697 47.8773 73.1078 84.8345 Freq. 98.4224 124.4890 136.2177 95.6342 125.7636 137.2774 Freq. 165.7738 175.8768 179.7743 165.2061 172.5390 179.1831 Freq. 208.6013 232.9612 255.1675 192.2090 204.8940 232.4310 Freq. 276.9068 309.9635 333.5209 254.8582 275.3390 306.3231 Freq. 355.0426 372.1401 400.6823 329.6301 351.8134 370.6041 Freq. 407.3848 421.1787 427.3598 398.9503 405.4726 421.4641 Freq. 433. 0242 460.9385 469.7289 429.6147 451.2325 468.5713 Freq. 494.9068 524.9670 560.3102 488.1846 520.7525 548.8198 Freq. 570. 2612 593.2115 608.2069 564.2778 566.2329 588.6715 Freq. 621.7065 646.2295 676.6275 601.0108 611.4715 639.1773 Freq. 703.4667 755.5010 761.1224 670.4855 679.7714 740.0584 Freq. 782.6368 800.0473 833.3656 756.8266 778.8766 781.4656 Freq. 876.5887 888.9391 903.3107 817.7921 831.4565 854.5214 Freq. 914.8855 929.0898 933.1824 888.9358 897.7032 904.0037 Freq. 946.1774 975.7880 986.1622 936.5494 954.3470 957.8729 Freq. 1006.316 2 1012.3429 1023.0808 964.6653 994.1672 1009.1591 Freq. 1031.191 2 1035.0950 1043.0197 1020.3256 1024.6609 1031.1776 Freq. 1049.578 9 1075.3149 1083.0956 1035.1955 1042.8636 1074.3430 Freq. 1140.008 6 1147.9555 1153.9013 1082.0809 1089.4312 1139.9334 Freq. 1164.205 3 1185.4796 1197.2828 1147.9286 1157.6984 1169.7163 Freq. 1202.891 8 1227.3076 1235.0154 1195.2761 1200.8898 1203.6054 Freq. 1264.181 5 1270.4631 1276.9361 1230.7527 1250.4426 1268.3951 Freq. 1285.836 2 1311.1026 1326.7683 1276.4712 1282.0772 1290.0491 Freq. 1335.892 5 1343.1894 1346.9874 1313.9371 1327.1092 1335.9328 Freq. 1370.200 8 1394.7606 1405.5416 1344.2979 1378.0256 1395.9757 Freq. 1418.218 0 1435.0830 1456.4757 1408.7028 1434.7233 1436.9083 Freq. 1465.922 6 1468.1477 1480.3082 1455.0455 1463.4090 1466.4532 Freq. 1493.051 0 1498.3574 1505.5311 1480.2400 1484.3281 1498.3001 Freq. 1517.344 4 1522.9947 1530.6495 1505.5526 1517.1817 1522.9219 Freq. 1534.559 1536.1446 1541.0093 1530.5690 1534.6626 1540.9140 20 2 Freq. 1564.640 8 1657.4856 1662.6829 1564.2889 1578.3237 1633.3561 Freq. 1688.105 4 1709.4886 1712.7025 1662.3766 1686.7712 1711.1220 Freq. 2340.655 4 3058.2563 3094.3445 2324.7851 3058.6631 3094.3652 Freq. 3101.799 7 3106.9627 3109.5533 3101.7936 3107.0909 3109.5362 Freq. 3113.947 7 3148.3012 3185.5426 3114.0086 3148.3435 3185.4626 Freq. 3197.588 2 3207.1858 3220.6987 3197.5761 3207.2010 3220.7420 Freq. 3221.544 5 3224.4719 3227.5479 3221.6024 3227.4392 3229.6863 Freq. 3229.792 0 3235.4322 3254.0122 3253.7311 3258.5328 3339.9527 Freq. 3258.604 3 3266.4312 3340.1049 3356.8421 3786.0417 3811.9142 Cit_307_b GS TS Freq. 16.1364 26.9805 32.4295 -69.3822 14.0235 30.2933 Freq. 47.4717 50.1475 68.3656 33.1018 44.5578 67.8234 Freq. 82.1814 107.1447 116.0468 79.7099 106.0690 113.6644 Freq. 142.5262 150.4329 200.4161 141.7295 148.8241 199.9098 Freq. 211.3583 217.9167 228.3425 210.8888 214.8056 222.8945 Freq. 247.1129 259.0930 287.8659 246.5852 255.4025 287.4496 Freq. 315.8010 353.1038 371.8895 315.4508 352.4869 371.4353 Freq. 391.5797 409.9512 419.2295 391.5430 409.2667 410.8409 Freq. 421.2875 443.6989 475.0224 419.3688 443.4646 475.1678 Freq. 511.6481 524.9183 556.1339 510.6193 523.2333 556.0964 Freq. 580.7457 608.5729 631.7576 580.6847 608.9805 632.4147 Freq. 640.1234 667.0845 683.2097 643.6360 667.7722 686.9076 Freq. 727.7160 741.1281 762.6175 727.9880 748.9463 763.0865 Freq. 772.7642 785.6686 809.5074 773.1251 786.9210 820.1901 Freq. 825.8018 845.1920 901.8788 844.8675 865.2841 902.3616 Freq. 909.5773 931.6581 954.7438 931.7710 940.9818 955.6031 Freq. 961.0445 968.4124 983.7998 961.3581 970.0543 984.2910 Freq. 993.4643 1022.4504 1028.0568 992.6332 1022.4579 1031.8631 Freq. 1040.608 8 1050.8663 1053.2925 1040.7221 1052.8047 1065.1573 Freq. 1065.152 9 1069.3216 1071.5197 1071.5601 1080.2140 1093.2645 Freq. 1072.415 9 1083.6182 1093.4272 1111.2785 1117.9497 1123.4442 Freq. 1111.794 0 1122.2669 1136.1426 1125.1857 1151.5528 1154.4548 Freq. 1174.161 1186.2954 1194.7231 1174.4995 1186.2817 1195.2211 21 2 Freq. 1216.840 3 1218.7292 1223.7023 1216.7791 1223.8693 1249.5266 Freq. 1237.182 6 1254.0292 1269.1902 1260.8033 1266.2489 1271.4134 Freq. 1271.485 4 1275.9536 1312.9418 1275.8074 1295.3565 1313.0703 Freq. 1321.216 3 1327.5484 1349.9735 1321.2786 1329.2137 1350.6652 Freq. 1355.985 6 1359.1922 1365.7700 1355.9925 1359.6052 1383.4781 Freq. 1385.622 3 1391.0993 1397.1326 1392.4434 1401.9445 1415.3599 Freq. 1415.339 5 1450.4954 1477.1006 1429.0408 1450.4940 1477.1760 Freq. 1481.270 7 1485.9031 1498.9802 1481.2607 1485.9480 1499.0032 Freq. 1502.306 7 1507.5401 1515.8561 1507.5279 1515.8797 1519.2328 Freq. 1519.250 3 1525.2774 1526.2064 1525.2499 1526.0946 1533.8653 Freq. 1533.854 8 1540.9903 1543.5483 1540.9920 1542.4321 1544.2018 Freq. 1546.493 0 1550.6070 1666.8692 1546.5035 1583.2994 1667.3822 Freq. 1676.504 4 1694.8941 1712.7320 1712.3225 1725.6256 1758.5687 Freq. 2339.770 7 3069.0125 3085.3185 2339.8407 2854.5791 2991.3011 Freq. 3091.249 8 3099.1928 3109.5275 3069.1157 3085.3084 3091.4020 Freq. 3113.075 9 3126.4500 3153.0470 3099.1752 3109.4574 3113.0550 Freq. 3173.560 0 3188.1555 3211.1785 3126.4576 3153.0525 3173.5442 Freq. 3219.971 9 3223.6068 3225.9908 3188.1701 3211.1742 3219.9274 Freq. 3232.701 1 3233.0359 3233.4312 3223.5857 3229.3606 3232.7095 Freq. 3233.908 3 3242.7928 3248.1192 3232.9847 3233.8471 3240.4998 Freq. 3253.301 3 3262.7195 3452.8642 3247.2865 3248.1664 3452.9535 Cit_282 GS TS Freq. 34.4813 34.7588 54.2607 -27.7295 23.7997 38.6676 Freq. 62.2344 85.2525 97.0564 50.5785 72.3309 89.3393 Freq. 121.5094 135.4867 148.8026 97.8094 111.4905 134.6540 22 Freq. 189.0736 213.2823 236.6790 175.6997 212.3226 236.1931 Freq. 245.7935 257.2552 303.6275 244.8737 255.1448 297.2336 Freq. 308.7529 324.4318 382.3859 307.0994 319.4776 379.7082 Freq. 408.9447 416.0740 428.3653 405.4416 415.2984 427.1245 Freq. 441.0725 456.1143 473.4780 435.3238 455.3592 471.9376 Freq. 520.5930 539.0900 547.2402 517.4180 538.2996 547.6028 Freq. 578.3026 598.1031 633.9007 573.7886 594.2665 634.1997 Freq. 643.3925 653.0981 699.3232 644.7402 653.2462 698.3998 Freq. 734.1514 744.8093 763.4841 734.6860 744.7793 762.9726 Freq. 775.1932 808.3343 844.9834 775.3827 807.5990 846.0526 Freq. 867.9318 884.6273 895.5402 868.6142 884.9561 895.6058 Freq. 916.0190 921.5169 927.7837 920.8816 925.2840 942.7067 Freq. 952.1589 966.3261 971.3520 956.0274 968.0271 970.7611 Freq. 1021.421 5 1033.9728 1043.4303 1021.7746 1034.1255 1043.8057 Freq. 1047.616 1 1053.5935 1075.8580 1048.2190 1072.8095 1079.6929 Freq. 1108.157 9 1140.3564 1147.4697 1108.4299 1140.7260 1147.8469 Freq. 1160.477 7 1165.8072 1193.0602 1160.5801 1167.4004 1189.0063 Freq. 1207.376 6 1214.0845 1258.0567 1207.2777 1214.2713 1255.6191 Freq. 1267.750 6 1288.6071 1303.2575 1267.4995 1288.9297 1301.8760 Freq. 1309.332 5 1325.2061 1325.4088 1306.1617 1325.4465 1334.7079 Freq. 1345.604 8 1352.6120 1362.3448 1347.1086 1354.5368 1364.7142 Freq. 1364.623 5 1372.5144 1389.6438 1373.0336 1376.6938 1405.8624 Freq. 1410.220 0 1455.9670 1463.7525 1423.4660 1455.9457 1464.1137 Freq. 1491.008 1 1498.5836 1513.8016 1484.8211 1498.6060 1513.7775 Freq. 1526.838 9 1532.5215 1538.8967 1526.8375 1532.5295 1538.9077 Freq. 1550.531 3 1571.1740 1664.3511 1556.5188 1571.3669 1678.6119 Freq. 1678.810 1 1698.6810 1715.0189 1698.6269 1707.8122 1726.2577 Freq. 2350.798 4 3040.0996 3058.8151 2404.9223 3040.1083 3058.6980 Freq. 3073.762 3 3117.1071 3137.1892 3073.7083 3117.0482 3125.5968 Freq. 3138.789 8 3167.4063 3176.5045 3137.2221 3138.7514 3167.3276 23 Freq. 3219.602 4 3228.2295 3241.5233 3176.5137 3207.7354 3219.6170 Freq. 3245.303 5 3250.5433 3258.3102 3242.7493 3245.2686 3251.0348 Freq. 3267.148 3 3283.6476 3694.0993 3267.3907 3283.6558 3694.3224 Cit_261 GS TS Freq. 45.1876 56.3213 63.3114 -5.0835 55.1180 64.2086 Freq. 79.5935 109.4738 119.1819 79.4123 108.2886 120.0854 Freq. 123.5833 174.1984 186.6619 124.3995 173.7950 184.6828 Freq. 233.4986 236.2056 272.1040 231.1707 237.2100 272.3145 Freq. 280.9148 342.9606 375.9183 280.7068 339.8458 368.6761 Freq. 392.0500 417.3655 421.5721 375.7004 408.0877 416.4587 Freq. 434.2277 466.0517 503.8133 432.7226 441.1605 501.8342 Freq. 527.8517 548.1487 570.4495 522.1551 547.8900 569.1815 Freq. 597.5753 639.2788 648.0791 595.5130 630.1357 639.3033 Freq. 670.6413 686.1395 697.8792 667.0165 673.8659 692.9522 Freq. 753.9791 760.9933 801.6218 730.7677 744.8534 764.8799 Freq. 835.0035 867.0229 882.3346 831.1984 834.0521 840.5909 Freq. 895.7837 905.9238 914.7812 883.5032 885.9986 898.0424 Freq. 923.8805 964.6752 980.9258 909.9460 922.1639 948.4741 Freq. 983.9921 1027.1556 1034.3433 978.8169 1006.1321 1028.4639 Freq. 1038.576 8 1047.8542 1055.8020 1034.6222 1039.0697 1047.4265 Freq. 1104.349 3 1146.8059 1156.4887 1103.1742 1105.8113 1145.5472 Freq. 1179.301 3 1192.8119 1205.3135 1148.2783 1161.3424 1202.7847 Freq. 1232.415 6 1253.4694 1274.2350 1209.6668 1232.9280 1256.1443 Freq. 1294.160 8 1335.5324 1350.3386 1293.7534 1313.3356 1335.0698 Freq. 1360.084 5 1368.2664 1406.2516 1347.1392 1357.7493 1387.6333 Freq. 1416.461 7 1439.5202 1463.1410 1404.1701 1433.5891 1439.8978 Freq. 1475.805 8 1488.6784 1500.7942 1472.2252 1488.8480 1493.4525 Freq. 1516.469 2 1536.7529 1573.4998 1506.0234 1513.2136 1562.3159 Freq. 1596.605 9 1639.5286 1654.6405 1572.9808 1593.6876 1639.1984 Freq. 1677.025 0 1690.1478 2354.7684 1675.6793 1688.7389 2344.8669 Freq. 3058.014 2 3123.4770 3189.6892 3058.0102 3123.4204 3189.4666 Freq. 3207.633 3230.9902 3232.9129 3207.6040 3231.2965 3233.6050 24 5 Freq. 3236.794 4 3245.8181 3261.4685 3261.3978 3272.1074 3276.8891 Freq. 3262.065 0 3271.7542 3276.6364 3288.3975 3514.5167 3665.4169 25