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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