Shortened validation procedure for a method of quantitative analysis of 45 amino acids in plasma on tandem mass spectrometer Marija Zekušić1, Ana Škaričić1, Ksenija Fumić1, Karmen Bilić1, Dunja Rogić1, Danijela Petković-Ramadža2, Mario Ćuk2, Lidija Paležac2, Vladimir Sarnavka2, Ivo Barić2,3 Introduction High performance liquid chromatography, combined with tandem mass spectrometry (LC-MS/MS), is a technology used for quantitative measurement of amino acids in plasma, urine and cerebrospinal fluid. For analysis, only 40 µL of sample is needed to quantify 45 amino acids in a very short time span. Materials and methods The validation was carried out using aTRAQ™ reagent manufactured by AB Sciex. Amino acids were chromatographically separated on the AB Sciex C18 column (5 µm, 4.6 mm x 150 mm) on UPLC-NEXERA instrument manufactured by Shimadzu, with binary gradient of water (mobile phase A) and methanol (mobile phase B) and addition of 0.1% of formic acid and 0.01% of heptafluorobutyric acid to both mobile phases. Amino acids were quantified on API 3200 mass spectrometer manufactured by AB Sciex. Shortened validation procedure for the method included testing of between-run and within-run imprecision, inaccuracy, limit of detection, limit of quantitation and linearity. Aim Shortened validation procedure for a method of quantifying 45 amino acids in plasma. SIGMA within-run SIGMA between-run Amino Acid X SIGMA 250 SD CV UPPER LIMIT X+2SD LOWER LIMIT X-2SD   PSer 234 25,54 10,90 285 183 PEtN 229 18,35 8,03 265 192 Tau 274 13,04 4,75 301 248 Asn 184 12,19 6,63 208 160 Ser 290 16,04 5,53 322 258 Gly 262 13,40 5,11 289 235 Hyp 239 24,92 10,44 189 EtN 250 18,19 7,28 286 213 Gln 292 18,98 6,51 330 254 Asp 31,56 11,50 338 211 Cit 261 20,33 7,80 220 Thr 270 21,11 313 228 Sar 182 34,67 19,01 252 113 bAla 282 20,43 7,23 323 242 Ala 19,28 7,03 236 Glu 222 14,85 6,71 251 His 18,25 6,75 307 1MHis 18,71 7,36 217 3MHis 249 12,42 5,00 273 224 Ans 260 21,28 8,19 302 Car 232 20,99 9,05 190 Aad 15,17 6,54 201 GABA 280 15,44 5,51 311 bAib 271 15,13 5,59 241 Abu 17,53 6,42 308 238 Arg 15,18 5,63 300 Pro 226 32,43 14,38 161 Orn 268 17,70 6,60 304 233 Cth 257 20,14 7,85 297 216 Cys 185 16,06 8,68 153 Hyl 266 18,77 7,07 303 Lys 263 19,84 7,54 223 Met 16,64 6,70 215 Val 21,52 8,09 309 Nva 139 10,74 7,74 117 Tyr 17,56 231 Hcy 253 15,31 6,06 283 Ile 267 17,93 6,72 Leu 264 20,30 7,69 305 Nle 125 12,20 9,74 150 101 Phe 20,73 7,86 Trp 245 17,50 7,14 210 Amino Acid X SIGMA 250 SD CV   PSer 250 11,68 4,67 PEtN 222 15,33 6,89 Tau 266 18,15 6,83 Asn 176 9,86 5,6 Ser 281 17,07 6,07 Gly 258 13,5 5,23 Hyp 255 12,12 4,76 EtN 251 13,05 5,2 Gln 256 60,21 23,53 Asp 288 7,73 2,68 Cit 262 15,61 5,95 Thr 263 13,02 4,95 Sar 235 25,7 10,92 bAla 24,73 9,43 Ala 24,48 9,35 Glu 260 27,16 10,43 His 264 14,22 5,4 1MHis 239 16,85 7,05 3MHis 238 16,37 6,88 Ans 241 22,82 9,46 Car 223 11,54 5,17 Aad 231 9,42 4,08 GABA 271 16,06 5,91 bAib 257 15,04 5,86 Abu 269 13,23 4,93 Arg 20,92 8,17 Pro 13,61 5,3 Orn 11,89 4,51 Cth 17 6,77 Cys 192 13,57 7,06 Hyl 10,29 4 Lys 11,88 4,56 Met 18,36 6,99 Val 11,51 4,39 Nva 136 3,71 2,72 Tyr 12,14 4,66 Hcy 246 14,36 5,85 Ile 13,36 5,09 Leu 261 10,76 4,13 Nle 124 10,2 8,22 Phe Trp 17,27 6,63 SKML within-run SKML between-run Amino Acid Ref Range (µm) X SKML SD CV UPPER LIMIT (X+2SD) LOWER LIMIT (X-2SD)   Tau 156-218 186 6,50 3,49 199 173 Asn 51-148 97 4,85 4,98 107 88 Ser 115-171 131 5,60 4,28 142 120 Gly 413-615 530 24,95 4,71 580 480 Hyp 65-109 3,30 3,74 95 82 Gln 503-735 645 28,72 4,46 702 587 Asp 29-64 40 1,88 4,75 43 36 Cit 99-145 122 8,56 7,02 139 105 Thr 136-182 163 6,95 4,27 176 149 Ala 312-434 380 16,93 4,45 414 347 Glu 93-143 114 4,34 3,81 His 178-280 232 7,54 3,24 247 217 Abu 24-38 33 1,46 30 Arg 209-307 260 9,41 3,61 279 242 Pro 240-368 297 12,41 4,17 322 273 Orn 265-395 329 12,96 3,94 355 303 Cth 17-44 31 2,18 6,96 27 Cys 33-56 19 1,38 7,34 22 16 Lys 222-330 286 14,76 5,15 316 257 Met 198-294 222 8,94 4,02 240 204 Val 335-451 397 17,28 4,36 431 362 Tyr 185-283 236 8,68 3,68 254 219 Ile 155-225 190 10,45 5,49 211 169 Leu 239-341 298 14,06 4,72 326 270 Phe 291-417 16,44 4,63 388 Trp 75-122 90 3,67 4,09 Amino Acid Ref Range (µM) X SKML SD CV   Tau 156-218 175 9 5,1 Asn 51-148 91 2 2,7 Ser 115-171 125 6 Gly 413-615 498 24 4,8 Hyp 65-109 85 4 Gln 503-735 605 29 Asp 29-64 39 5 12,8 Cit 99-145 116 4,7 Thr 136-182 151 7 4,9 Ala 312-434 355 15 4,3 Glu 93-143 113 17 15,2 His 178-280 217 Abu 24-38 31 1 4,5 Arg 209-307 252 10 3,8 Pro 240-368 296 Orn 265-395 312 3,2 Cth 17-44 30 5,5 Cys 33-56 21 18,3 Lys 222-330 265 12 4,6 Met 198-294 234 20 8,4 Val 335-451 374 4,1 Tyr 185-283 224 4,0 Ile 155-225 181 3,7 Leu 239-341 277 11 Phe 291-417 339 14 4,2 Trp 75-122 94 7,2 Results Calculated values of the coefficient of variation (CV) for within-run and between-run imprecision and inaccuracy for most amino acids were lower than 20%, with the exception of glutamine (CV=24%). Limit of detection (LoD) and limit of quantitation (LoQ) were lower than 2 µmol/L for most amino acids except for serine, glycine and ß-alanine which had somewhat higher LoQ. The method was linear up to 1000 µmol/L for most amino acids. zekusicm@kbc-zagreb.hr