Figure 1Representative chromatograms of HPLC with OPA-labeled histamine. (A) Standard solutions of histamine and 3-methyl histamine (IS). (B) Histamine-added pharmaceuticals extracted by SDB-L SPE and (C) by SDB-X SPE.
Figure 2HPLC chromatograms of different fluorescent labeling agents: (A) OPA; and (B) NDA. Six different concentrations of histamine-containing samples with IS were analyzed and the chromatograms overlapped after each fluorescent agents’ derivatization.
Figure 3Comparison of analytic methods of histamine using 15 lots of histamine-added human immunoglobulin: current lot-release test method using fluorometry (red squares) and new HPLC method (blue diamonds) are shown. For HPLC method, n = 3; Bars, SD.
Figure 4Trend analysis of histamine content in 15 lots of human immunoglobulin preparations with histamine. Mean ± SD was 0.168 ± 0.016 μg/vial. 2SD and 4SD lines are shown as dotted lines.
Scheme 1Derivatization reaction schemes of OPA (A) and NDA (B) with histamine.
Table 1Effect of reduced extraction volume in SPE cartridge on HPLC analysis of histamine and internal standard in human immunoglobulin spiked with 0025 μg of histamine (n = 6)
Extraction volume |
Recovery (%) |
Histamine area |
IS area |
2 mL |
111.72–112.92 |
19.12–20.16 |
29.63–30.88 |
5 mL |
108.63–115.92 |
9.69–10.60 |
14.45–14.54 |
Table 2Recovery of histamine in matrix samples prepared by spiking different concentrations of histamine with 0.075 μg/mL of IS
Spiked amount (μg) |
Final conc. (μg/mL) |
HPLC analysis result (μg/mL) |
RSD (%) |
Recovery (%) |
0.05 |
0.025 |
0.0258 ± 0.0008 |
3.27 |
103.2 |
0.10 |
0.050 |
0.0490 ± 0.0019 |
3.85 |
97.9 |
0.15 |
0.075 |
0.0713 ± 0.0024 |
3.34 |
95.0 |
0.20 |
0.100 |
0.0916 ± 0.0017 |
1.89 |
91.6 |
0.30 |
0.150 |
0.1525 ± 0.0014 |
0.94 |
101.7 |
Table 3Intra-day and inter-day analyses of HPLC using NDA-labeled histamine in matrix sample prepared by spiking histamine with 0.075 μg/mL of IS
Histamine spiked (μg) |
Histamine concentration (μg/mL) |
Day |
Histamine conc. (μg/mL) |
RSD (%) |
Recovery (%) |
0.15 |
0.075 |
1 |
0.0747 ± 0.0004 |
0.51 |
99.59 |
0.15 |
0.075 |
2 |
0.0777 ± 0.0004 |
0.56 |
103.61 |
0.15 |
0.075 |
3 |
0.0784 ± 0.0011 |
1.40 |
104.47 |
Table 4Linearity of dose–response curves of HPLC analysis for NDA-labeled histamine on days 1–3
HPLC analysis |
R2
|
Dose response curve parameters
|
Slope |
Intercept |
Day 1 |
0.9950 |
0.0245 |
−0.1880 |
Day 2 |
0.9916 |
0.0243 |
−0.1930 |
Day 3 |
0.9928 |
0.0277 |
−0.0892 |
Mean ± SD |
0.9931 ± 0.0017 |
0.0255 ± 0.0019 |
−0.1567 ± 0.0585 |