1. 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
186
6,50
3,49
199
173
Asn
51-148 97
4,85
4,98
107 88
Ser
131
5,60
4,28
142
120
Gly
530
24,95
4,71
580
480
Hyp
65-109
3,30
3,74 95 82
Gln
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
163
6,95
4,27
176
149
Ala
380
16,93
4,45
414
347
Glu
93-143
114
4,34
3,81
His
232
7,54
3,24
247
217
Abu
24-38 33
1,46 30
Arg
260
9,41
3,61
279
242
Pro
297
12,41
4,17
322
273
Orn
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
286
14,76
5,15
316
257
Met
222
8,94
4,02
240
204
Val
397
17,28
4,36
431
362
Tyr
236
8,68
3,68
254
219
Ile
190
10,45
5,49
211
169
Leu
298
14,06
4,72
326
270
Phe
16,44
4,63
388
Trp
75-122 90
3,67
4,09
Amino Acid
Ref Range (µM) X
SKML SD CV
Tau
175 9
5,1
Asn
51-148 91 2
2,7
Ser
125 6
Gly
498 24
4,8
Hyp
65-109 85 4
Gln
605 29
Asp
29-64 39 5
12,8
Cit
99-145
116
4,7
Thr
151 7
4,9
Ala
355 15
4,3
Glu
93-143
113 17
15,2
His
217
Abu
24-38 31 1
4,5
Arg
252 10
3,8
Pro
296
Orn
312
3,2
Cth
17-44 30
5,5
Cys
33-56 21
18,3
Lys
265 12
4,6
Met
234 20
8,4
Val
374
4,1
Tyr
224
4,0
Ile
181
3,7
Leu
277 11
Phe
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.