Chinese Journal of Chromatography ›› 2021, Vol. 39 ›› Issue (3): 338-345.DOI: 10.3724/SP.J.1123.2020.06005
• Technical Notes • Previous Articles
FANG Li1,*(), QIU Fengmei2, YU Xinwei1
Received:
2020-06-02
Online:
2021-03-08
Published:
2021-02-03
Contact:
FANG Li
Supported by:
CLC Number:
FANG Li, QIU Fengmei, YU Xinwei. Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2021, 39(3): 338-345.
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URL: https://www.chrom-china.com/EN/10.3724/SP.J.1123.2020.06005
Step No. | Start time/ min | Step time/ min | Function | Loading pump | Eluting pump | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Flow rate/ (mL/min) | φ(0.1% FA)/% | φ(MeOH)/ % | φ(H2O)/ % | φ(ACN)/ % | Flow rate/ (mL/min) | φ(4 mmol/L CH3COONH4)/% | φ(MeOH)/ % | ||||||
1 | 0.0 | 1.0 | loading | 1.5 | 100 | 0 | 0 | 0 | 0.3 | 95 | 5 | ||
2 | 1.0 | 1.0 | transferring | 0.1 | 80 | 0 | 0 | 20 | 0.2 | 95 | 5 | ||
3 | 2.0 | 2.0 | washing | 1.5 | 0 | 0 | 90 | 10 | 0.3 | 80 | 20 | ||
4 | 4.0 | 1.5 | washing | 1.5 | 0 | 0 | 0 | 100 | 0.3 | 60 | 40 | ||
5 | 5.5 | 1.0 | washing | 1.5 | 0 | 100 | 0 | 0 | 0.3 | 40 | 60 | ||
6 | 6.5 | 0.5 | washing | 1.5 | 0 | 0 | 0 | 100 | 0.3 | 10 | 90 | ||
7 | 7.0 | 2.0 | filling the loop | 1.5 | 80 | 0 | 0 | 20 | 0.3 | 10 | 90 | ||
8 | 9.0 | 0.2 | equilibrating | 1.5 | 100 | 0 | 0 | 0 | 0.3 | 95 | 5 | ||
9 | 9.2 | 1.8 | equilibrating | 1.5 | 100 | 0 | 0 | 0 | 0.3 | 95 | 5 |
Table 1 Gradient elution procedures of online clean-up and chromatographic separation
Step No. | Start time/ min | Step time/ min | Function | Loading pump | Eluting pump | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Flow rate/ (mL/min) | φ(0.1% FA)/% | φ(MeOH)/ % | φ(H2O)/ % | φ(ACN)/ % | Flow rate/ (mL/min) | φ(4 mmol/L CH3COONH4)/% | φ(MeOH)/ % | ||||||
1 | 0.0 | 1.0 | loading | 1.5 | 100 | 0 | 0 | 0 | 0.3 | 95 | 5 | ||
2 | 1.0 | 1.0 | transferring | 0.1 | 80 | 0 | 0 | 20 | 0.2 | 95 | 5 | ||
3 | 2.0 | 2.0 | washing | 1.5 | 0 | 0 | 90 | 10 | 0.3 | 80 | 20 | ||
4 | 4.0 | 1.5 | washing | 1.5 | 0 | 0 | 0 | 100 | 0.3 | 60 | 40 | ||
5 | 5.5 | 1.0 | washing | 1.5 | 0 | 100 | 0 | 0 | 0.3 | 40 | 60 | ||
6 | 6.5 | 0.5 | washing | 1.5 | 0 | 0 | 0 | 100 | 0.3 | 10 | 90 | ||
7 | 7.0 | 2.0 | filling the loop | 1.5 | 80 | 0 | 0 | 20 | 0.3 | 10 | 90 | ||
8 | 9.0 | 0.2 | equilibrating | 1.5 | 100 | 0 | 0 | 0 | 0.3 | 95 | 5 | ||
9 | 9.2 | 1.8 | equilibrating | 1.5 | 100 | 0 | 0 | 0 | 0.3 | 95 | 5 |
Analyte | Scan mode | Precursor ion (m/z) | Product ion (m/z) | S-lens/ V | CE/ eV |
---|---|---|---|---|---|
α-Amanitin | ESI+ | 919.6 | 259.0*/86.0 | 180 | 33/48 |
β-Amanitin | ESI+ | 920.6 | 259.0*/86.0 | 180 | 32/46 |
γ-Amanitin | ESI+ | 903.6 | 243.0*/86.0 | 180 | 37/54 |
Phallacidin | ESI+ | 847.6 | 157.0*/329.9 | 160 | 50/28 |
Phalloidin | ESI+ | 789.6 | 157.0*/330.0 | 160 | 45/33 |
Table 2 Detection parameters of amanita peptide toxins
Analyte | Scan mode | Precursor ion (m/z) | Product ion (m/z) | S-lens/ V | CE/ eV |
---|---|---|---|---|---|
α-Amanitin | ESI+ | 919.6 | 259.0*/86.0 | 180 | 33/48 |
β-Amanitin | ESI+ | 920.6 | 259.0*/86.0 | 180 | 32/46 |
γ-Amanitin | ESI+ | 903.6 | 243.0*/86.0 | 180 | 37/54 |
Phallacidin | ESI+ | 847.6 | 157.0*/329.9 | 160 | 50/28 |
Phalloidin | ESI+ | 789.6 | 157.0*/330.0 | 160 | 45/33 |
Fig. 3 Effect of online purification conditions on peak intensities of amanita peptide toxins (n=3) a. loading solution; b. eluting solution; c. transfer flow; d. transfer time.
Toxin | Linear range/(μg/L) | Solvent | Urine | |||||||
---|---|---|---|---|---|---|---|---|---|---|
Curve | R2 | Curve | R2 | ME/% | LOD/(μg/L) | LOQ/(μg/L) | ||||
α-Amanitin | 1.0-50.0 | y=918.615x -191.188 | 0.9994 | y=578.167x-298.264 | 0.9990 | -37.1 | 0.3 | 1.0 | ||
β-Amanitin | 1.0-50.0 | y=1018.51x-34.4314 | 0.9970 | y=481.957x-61.4861 | 0.9998 | -52.7 | 0.3 | 1.0 | ||
γ-Amanitin | 1.0-50.0 | y=1371.64x -126.402 | 0.9996 | y=915.107x-172.278 | 0.9990 | -33.3 | 0.3 | 1.0 | ||
Phallacidin | 1.0-50.0 | y=1794.66x-587.165 | 0.9994 | y=942.94x-126.634 | 0.9999 | -47.5 | 0.3 | 1.0 | ||
Phalloidin | 1.0-50.0 | y=4240.93x+3.11143 | 0.9992 | y=2899.95x+548.874 | 0.9995 | -31.6 | 0.15 | 0.5 |
Table 3 Regression equations, correlation coefficients, matrix effects, LODs, and LOQs of the five amanita peptide toxins
Toxin | Linear range/(μg/L) | Solvent | Urine | |||||||
---|---|---|---|---|---|---|---|---|---|---|
Curve | R2 | Curve | R2 | ME/% | LOD/(μg/L) | LOQ/(μg/L) | ||||
α-Amanitin | 1.0-50.0 | y=918.615x -191.188 | 0.9994 | y=578.167x-298.264 | 0.9990 | -37.1 | 0.3 | 1.0 | ||
β-Amanitin | 1.0-50.0 | y=1018.51x-34.4314 | 0.9970 | y=481.957x-61.4861 | 0.9998 | -52.7 | 0.3 | 1.0 | ||
γ-Amanitin | 1.0-50.0 | y=1371.64x -126.402 | 0.9996 | y=915.107x-172.278 | 0.9990 | -33.3 | 0.3 | 1.0 | ||
Phallacidin | 1.0-50.0 | y=1794.66x-587.165 | 0.9994 | y=942.94x-126.634 | 0.9999 | -47.5 | 0.3 | 1.0 | ||
Phalloidin | 1.0-50.0 | y=4240.93x+3.11143 | 0.9992 | y=2899.95x+548.874 | 0.9995 | -31.6 | 0.15 | 0.5 |
Toxin | Spiked level/ (μg/L) | Intra-day (n=6) | Inter-day (n=3) | |||
---|---|---|---|---|---|---|
Recovery/ % | RSD/ % | Recovery/ % | RSD/ % | |||
α-Amanitin | 2.0 | 101.5 | 9.8 | 107.1 | 5.5 | |
5.0 | 100.5 | 7.4 | 106.0 | 4.3 | ||
10.0 | 102.9 | 5.6 | 104.4 | 1.9 | ||
β-Amanitin | 2.0 | 95.4 | 14.2 | 97.3 | 5.5 | |
5.0 | 89.2 | 4.0 | 94.4 | 4.3 | ||
10.0 | 99.2 | 7.8 | 94.6 | 4.6 | ||
γ-Amanitin | 2.0 | 108.6 | 13.6 | 112.7 | 7.2 | |
5.0 | 98.1 | 7.7 | 100.4 | 2.5 | ||
10.0 | 95.3 | 6.4 | 96.7 | 2.9 | ||
Phallacidin | 2.0 | 96.0 | 14.5 | 93.5 | 5.6 | |
5.0 | 91.7 | 6.5 | 89.4 | 5.7 | ||
10.0 | 93.0 | 4.0 | 87.5 | 4.0 | ||
Phalloidin | 2.0 | 90.2 | 5.7 | 87.9 | 2.3 | |
5.0 | 87.0 | 2.7 | 86.8 | 2.1 | ||
10.0 | 89.1 | 4.0 | 88.0 | 0.8 |
Table 4 Recovery and precision of the method
Toxin | Spiked level/ (μg/L) | Intra-day (n=6) | Inter-day (n=3) | |||
---|---|---|---|---|---|---|
Recovery/ % | RSD/ % | Recovery/ % | RSD/ % | |||
α-Amanitin | 2.0 | 101.5 | 9.8 | 107.1 | 5.5 | |
5.0 | 100.5 | 7.4 | 106.0 | 4.3 | ||
10.0 | 102.9 | 5.6 | 104.4 | 1.9 | ||
β-Amanitin | 2.0 | 95.4 | 14.2 | 97.3 | 5.5 | |
5.0 | 89.2 | 4.0 | 94.4 | 4.3 | ||
10.0 | 99.2 | 7.8 | 94.6 | 4.6 | ||
γ-Amanitin | 2.0 | 108.6 | 13.6 | 112.7 | 7.2 | |
5.0 | 98.1 | 7.7 | 100.4 | 2.5 | ||
10.0 | 95.3 | 6.4 | 96.7 | 2.9 | ||
Phallacidin | 2.0 | 96.0 | 14.5 | 93.5 | 5.6 | |
5.0 | 91.7 | 6.5 | 89.4 | 5.7 | ||
10.0 | 93.0 | 4.0 | 87.5 | 4.0 | ||
Phalloidin | 2.0 | 90.2 | 5.7 | 87.9 | 2.3 | |
5.0 | 87.0 | 2.7 | 86.8 | 2.1 | ||
10.0 | 89.1 | 4.0 | 88.0 | 0.8 |
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