Table 5 Evaluation of samples at 3 concentration levels for estimation of stability and recovery of the UHPLC–MS method
From: Determination of immunogenic proteins in biopharmaceuticals by UHPLC–MS amino acid analysis
Freeze–thaw stability (3 cycles) | Autosampler stability (48 h) | Recovery | ||||
|---|---|---|---|---|---|---|
Concentration found (µM) | Accuracy (%RE) | Concentration found (µM) | Accuracy (%RE) | Found concentration (µM) | Recovery (%) | |
Glycine | ||||||
5 µM | 5.20 | 4.00 | 5.25 | 5.00 | 4.70 | 94.0 |
70 µM | 69.15 | − 1.21 | 72.80 | 4.00 | 64.15 | 91.6 |
300 µM | 302.70 | 0.90 | 299.05 | − 0.30 | 288.40 | 96.1 |
Alanine | ||||||
5 µM | 5.35 | 7.00 | 4.90 | − 2.00 | 4.70 | 94.0 |
70 µM | 71.35 | 1.93 | 66.80 | − 4.57 | 70.55 | 100.8 |
300 µM | 300.65 | 0.22 | 299.15 | − 0.28 | 296.75 | 98.9 |
Serine | ||||||
5 µM | 5.10 | 2.00 | 4.85 | − 3.00 | 4.60 | 92.0 |
70 µM | 65.55 | − 6.36 | 69.05 | − 1.36 | 68.00 | 97.1 |
300 µM | 297.05 | − 0.98 | 304.80 | 1.60 | 298.10 | 99.4 |
Proline | ||||||
5 µM | 5.10 | 2.00 | 4.90 | − 2.00 | 4.70 | 94.0 |
70 µM | 71.15 | 1.64 | 70.30 | − 0.43 | 69.60 | 99.4 |
300 µM | 303.90 | 1.30 | 293.00 | − 2.33 | 296.80 | 98.9 |
Valine | ||||||
5 µM | 5.20 | 4.00 | 5.00 | 0.00 | 5.05 | 101.0 |
70 µM | 70.70 | 1.00 | 67.35 | − 3.79 | 68.75 | 98.2 |
300 µM | 304.45 | 1.48 | 300.65 | 0.22 | 295.65 | 98.6 |
Threonine | ||||||
5 µM | 4.95 | − 1.00 | 4.90 | − 2.00 | 4.95 | 99.0 |
70 µM | 68.35 | − 2.36 | 67.95 | − 2.93 | 67.45 | 96.36 |
300 µM | 305.75 | 1.92 | 301.30 | 0.43 | 299.85 | 99.9 |
Cysteine | ||||||
2.5 µM | 2.43 | − 2.80 | 2.43 | − 2.80 | 2.40 | 96.0 |
35 µM | 34.23 | − 2.20 | 33.15 | − 5.29 | 33.98 | 97.1 |
150 µM | 148.50 | − 1.00 | 148.70 | − 0.87 | 141.73 | 94.5 |
Isoleucine | ||||||
5 µM | 5.05 | 1.00 | 4.95 | − 1.00 | 5.00 | 100.0 |
70 µM | 71.40 | 2.00 | 70.65 | 0.93 | 71.20 | 101.7 |
300 µM | 302.45 | 0.82 | 305.40 | 1.80 | 302.20 | 100.7 |
Leucine | ||||||
5 µM | 5.10 | 2.00 | 4.95 | − 1.00 | 5.00 | 100.0 |
70 µM | 70.95 | 1.36 | 70.55 | 0.79 | 71.95 | 102.8 |
300 µM | 305.85 | 1.95 | 299.10 | − 0.30 | 302.30 | 100.8 |
Asparagine | ||||||
5 µM | 4.95 | − 1.00 | 4.80 | − 4.00 | 4.70 | 94.0 |
70 µM | 71.95 | 2.79 | 68.10 | − 2.71 | 67.60 | 96.6 |
300 µM | 287.85 | − 4.05 | 296.55 | − 1.15 | 292.20 | 97.4 |
Aspartic acid | ||||||
5 µM | 5.30 | 6.00 | 4.85 | − 3.00 | 4.85 | 97.0 |
70 µM | 67.80 | − 3.14 | 67.45 | − 3.64 | 63.40 | 90.6 |
300 µM | 305.3 | 1.77 | 307.70 | 2.57 | 292.15 | 97.4 |
Glutamine | ||||||
5 µM | 4.75 | − 5.00 | 4.95 | − 1.00 | 4.65 | 93.0 |
70 µM | 71.15 | 1.64 | 68.15 | − 2.64 | 67.35 | 96.2 |
300 µM | 295.15 | − 1.62 | 297.85 | − 0.72 | 287.85 | 96.0 |
Lysine | ||||||
5 µM | 5.0 | 0.00 | 4.85 | − 3.00 | 4.75 | 95.0 |
70 µM | 69.25 | − 1.07 | 67.20 | − 4.00 | 68.15 | 97.4 |
300 µM | 309.45 | 3.15 | 287.35 | − 4.22 | 285.20 | 95.1 |
Glutamic acid | ||||||
5 µM | 4.75 | − 5.00 | 4.90 | − 2.00 | 4.80 | 96.0 |
70 µM | 65.50 | − 6.43 | 68.10 | − 2.71 | 70.20 | 100.3 |
300 µM | 298.35 | − 0.55 | 300.70 | 0.23 | 294.35 | 98.1 |
Methionine | ||||||
5 µM | 4.75 | − 5.00 | 5.00 | 0.00 | 4.95 | 99.0 |
70 µM | 69.70 | − 0.43 | 67.45 | − 3.64 | 66.30 | 94.7 |
300 µM | 294.90 | − 1.70 | 299.50 | − 0.17 | 295.20 | 98.4 |
Histidine | ||||||
5 µM | 4.40 | − 12.00 | 4.85 | − 3.00 | 4.50 | 90.0 |
70 µM | 71.70 | 2.43 | 67.10 | − 4.14 | 64.65 | 92.4 |
300 µM | 297.20 | − 0.93 | 303.90 | 1.30 | 295.70 | 98.6 |
Phenylalanine | ||||||
5 µM | 4.90 | − 2.00 | 4.80 | − 4.00 | 4.85 | 97.0 |
70 µM | 69.95 | − 0.07 | 67.45 | − 3.64 | 65.55 | 93.6 |
300 µM | 306.10 | 2.03 | 304.95 | 1.65 | 292.75 | 97.6 |
Arginine | ||||||
5 µM | 5.0 | 0.00 | 4.90 | − 2.00 | 5.00 | 100.0 |
70 µM | 70.05 | 0.07 | 69.25 | − 1.07 | 69.15 | 98.8 |
300 µM | 297.55 | − 0.82 | 301.70 | 0.57 | 294.95 | 98.3 |
Tyrosine | ||||||
5 µM | 4.85 | − 3.00 | 4.85 | − 3.00 | 5.00 | 100.0 |
70 µM | 70.15 | 0.21 | 66.70 | − 4.71 | 67.70 | 96.7 |
300 µM | 299.95 | − 0.02 | 306.50 | 2.17 | 297.70 | 99.2 |
Tryptophan | ||||||
5 µM | 5.10 | 2.00 | 5.05 | 1.00 | 5.05 | 101.0 |
70 µM | 70.80 | 1.14 | 67.80 | − 3.14 | 71.35 | 101.9 |
300 µM | 298.50 | − 0.50 | 297.80 | − 0.73 | 294.85 | 98.3 |