We used the same pooled plasma of feminine EHBRs treated with GA for 12 orally?h as inside our previous research18

We used the same pooled plasma of feminine EHBRs treated with GA for 12 orally?h as inside our previous research18. Assay of binding of substances 2 and 3 to serum albumin We measured the binding ratios of substances 2 and 3 to serum albumin with the same process using pooled plasma of feminine EHBRs as inside our previous research18. Patient experiencing licorice-induced pseudoaldosteronism The individual was a 76-year-old female. was isolated in the bile of rats treated 1 first.0, MeOH)] exhibited a deprotonated molecule in 565 (M-H)? in the ESIMS, as well as the molecular formulation, C30H46O8S, was established by HRESIMS [565.2835, (M-H)?, 4?C group by Students mock cells by Dunnetts multiple Mrp2, where the concentrations of 2 and 1 in the bile of SD rats injected enterohepatic circulation. This was supported by the biphasic profile of plasma GA concentration in SD rats orally treated with GA. Finally, GA would be eliminated into the feces as GA or 3. On the other hand, the concentrations of 3MGA in the plasma and the urine of EHBRs orally treated with GA were much lower than those of 1 1, 2, and 3, exposing that 3MGA is usually a minor metabolite of GA in EHBRs. We found that most of 2 and 3 in plasma was bound to albumin, suggesting that these two compounds would not be eliminated into the urine glomerular filtration, but rather tubular secretion. As 1 experienced a high binding ratio to albumin and was a substrate for OAT1 and 318 and 2 and 3 share a high binding ratio to albumin, these compounds can be transported into the cells actively OAT1 and 3, and eliminated into the urine by tubular secretion. As GA also has a high binding ratio to albumin but is not a substrate for OATs, GA was not detected in the urine in either SD rats or EHBRs. The IC50 values of GL and its metabolites on 11because 111.0, MeOH); UV (MeOH) 6289) nm; ECD (MeOH) (565 [M-H]?; HRESIMS 565.2835 [M-H]? (calcd for C30H45O8S, 565.2835). 18(5.58 (1H, s, H-12), 3.95 (1H, dd 12.0, 4.5?Hz, H-3), 2.73 (1H, brd 13.5?Hz, H-1a), 2.48 (1H, s, H-9), 2.20 (1H, brd 14.5?Hz, H-18), 2.14 (1H, dd 14.0, 4.5?Hz, H-16a), 2.07 (1H, m, H-2a), 1.94 (1H, m, H-21a), 1.87 (1H, m, H-15a), 1.85 (1H, m, H-19a), 1.81 (1H, m, H-2b), 1.75 (1H, m, H-7a), 1.72 (1H, m, H-19b), 1.65 (1H,brd 13.0?Hz, H-6a), 1.60 (1H, m, H-6b), 1.45 (1H, m, H-7b), 1.44 (3H, s, H-27), 1.41 (1H, m, H-21b), 1.40 (2H, m, H-22), 1.25 (1H, brd 12.5?Hz, H-15b), 1.17 (3H, s, H-29), 1.16 (3H, s, H-25), 1.15 (3H, s, H-26), 1.06 (3H, s, H-23), 1.05 (1H, m, H-1b), 1.04 (1H, m, H-16b), 0.87 (1H, m, H-5), 0.86 (3H, s, H-24), 0.84 (3H, s, H-28) and 13C-NMR (CD3OD, 125?MHz) 202.6 (C-11), 180.5 (C-30), 172.9 (C-13), 128.9 (H-12), 87.2 (C-3), 63.0 (C-9), 56.6 (C-5), 49.9 (C-18), 46.7 (C-8), 44.9 (C-20), 44.6 (C-14), 42.4 (C-19), 40.1 (C-1), 39.9 (C-4), 39.0 (C-22), 38.2 (C-10), 33.8 (C-7), 33.0 (C-17), 32.0 (C-21), 29.2 (C-28), 28.7 (C-23), 28.7 (C-29), 27.6 (C-15), 27.4 (C-16), 25.2 (C-2), 23.8 (C-27), 19.3 (C-26), 18.6 (C-6), 17.0 (C-25), 16.9 (C-24); ESIMS 549 [M-H]-; HRESIMS 549.2888 [M-H]- (calcd for C30H45O7S, 549.2886). Determination of 11for 7?min), the concentrations of compounds 1, 2, 3, 3MGA, GA, and GL in the supernatant of the samples prepared from plasma and urine were measured using LC-ESIMS/MS under the following conditions: column, Scherzo SM-C18 (3?m, 3?mm for compound 1 (5.5?min), ESI(?) 565.5 to 96.5?for 2 (6.2?min), ESI(?) 549.5 to 96.5?for 3 (8.1?min), ESI(+) 647.6 to 453.6?for 3MGA (9.2?min), ESI(+) 471.3 to 91.0?for GA (8.8?min), ESI(+) 823.5 to 453.6?for GL (10.8?min), and ESI(+) 785.4 to 143.0?for astragaloside IV (3.0?min). Linear regressions over the concentration range of 32?nM to 20?M for each compound were examined.Other authors declare no competing financial and/or non-financial interests in relation to this work. Footnotes Publishers notice: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Kanichiro Ishiuchi and Osamu Morinaga contributed equally.. was collected and pooled. From 1 liter of this urine, we isolated 2 (1.9?mg) as a new GA metabolite, and 3 (0.4?mg) that was first isolated from your bile of rats treated 1.0, MeOH)] exhibited a deprotonated molecule at 565 (M-H)? in the ESIMS, and the molecular formula, C30H46O8S, was established by HRESIMS [565.2835, (M-H)?, 4?C group by Students mock cells by Dunnetts multiple Mrp2, where the concentrations of 2 and 1 in the bile of SD rats injected enterohepatic circulation. This was supported by the biphasic profile of plasma GA concentration in SD rats orally treated with GA. Finally, GA would be eliminated into the feces as GA or 3. On the other hand, the concentrations of 3MGA in the plasma and the urine of EHBRs Toloxatone orally treated with GA were much lower than those of 1 1, 2, and 3, exposing that 3MGA is usually a minor metabolite of GA in EHBRs. We found that most of 2 and 3 in plasma was bound to albumin, suggesting that these two compounds would not be eliminated into the urine glomerular filtration, but rather tubular secretion. As 1 experienced a high binding ratio to albumin and was a substrate for OAT1 and 318 and 2 and 3 share a high binding ratio to albumin, these compounds can be transported into the cells actively OAT1 and 3, and eliminated into the urine by tubular secretion. As GA also has a high binding ratio to albumin but is not a substrate for OATs, GA was not detected in the urine in either SD rats or EHBRs. The IC50 values of GL and its metabolites on 11because 111.0, MeOH); UV (MeOH) 6289) nm; ECD (MeOH) (565 [M-H]?; HRESIMS 565.2835 [M-H]? (calcd for C30H45O8S, 565.2835). 18(5.58 (1H, s, H-12), 3.95 (1H, dd 12.0, 4.5?Hz, H-3), 2.73 (1H, brd 13.5?Hz, H-1a), 2.48 (1H, s, H-9), 2.20 (1H, brd 14.5?Hz, H-18), 2.14 (1H, dd 14.0, 4.5?Hz, H-16a), 2.07 (1H, m, H-2a), 1.94 (1H, m, H-21a), 1.87 (1H, m, H-15a), 1.85 (1H, m, H-19a), 1.81 (1H, m, H-2b), 1.75 (1H, m, H-7a), 1.72 (1H, m, H-19b), 1.65 (1H,brd 13.0?Hz, H-6a), 1.60 (1H, m, H-6b), 1.45 (1H, m, H-7b), 1.44 (3H, s, H-27), 1.41 (1H, m, H-21b), 1.40 (2H, m, H-22), 1.25 (1H, brd 12.5?Hz, H-15b), 1.17 (3H, s, H-29), 1.16 (3H, s, H-25), 1.15 (3H, s, H-26), Toloxatone 1.06 (3H, s, H-23), 1.05 (1H, m, H-1b), 1.04 (1H, m, H-16b), 0.87 (1H, m, H-5), 0.86 (3H, s, H-24), 0.84 (3H, s, H-28) and 13C-NMR (CD3OD, 125?MHz) 202.6 (C-11), 180.5 (C-30), 172.9 (C-13), 128.9 (H-12), 87.2 (C-3), 63.0 (C-9), 56.6 (C-5), 49.9 (C-18), 46.7 (C-8), 44.9 (C-20), 44.6 (C-14), 42.4 (C-19), 40.1 (C-1), 39.9 (C-4), 39.0 (C-22), 38.2 (C-10), 33.8 (C-7), 33.0 (C-17), 32.0 (C-21), 29.2 (C-28), 28.7 (C-23), 28.7 (C-29), 27.6 (C-15), 27.4 (C-16), 25.2 (C-2), 23.8 (C-27), 19.3 (C-26), 18.6 (C-6), 17.0 (C-25), 16.9 (C-24); ESIMS 549 [M-H]-; HRESIMS 549.2888 [M-H]- (calcd for C30H45O7S, 549.2886). Determination of 11for 7?min), the concentrations of compounds 1, 2, 3, 3MGA, GA, and GL in the supernatant of the samples prepared from plasma and urine were measured using LC-ESIMS/MS under the following conditions: column, Scherzo SM-C18 (3?m, 3?mm for compound 1 (5.5?min), ESI(?) 565.5 to 96.5?for 2 (6.2?min), ESI(?) 549.5 to 96.5?for 3 (8.1?min), ESI(+) 647.6 to 453.6?for 3MGA (9.2?min), ESI(+) 471.3 to 91.0?for GA (8.8?min), ESI(+) 823.5 to 453.6?for GL (10.8?min), and ESI(+) 785.4 to 143.0?for astragaloside IV (3.0?min). Linear regressions over the concentration range of 32?nM to 20?M for each compound were examined using the peak-area ratio of the compounds to their internal requirements and the least-squares method ( em r /em 2? ?0.98). Uptake of.Finally, the membrane was washed twice with T-PBS and once with PBS, and exposed to 1?mg/ml of freshly prepared 4-chloro-1-naphthol-0.03% H2O2 in PBS for 15?min at room temperature. administered 1?mg/ml GA drinking water for 3 months, and their urine was collected and pooled. From 1 liter of this urine, we isolated 2 (1.9?mg) as a new GA metabolite, and 3 (0.4?mg) that was first isolated from your bile of rats treated 1.0, MeOH)] exhibited a deprotonated molecule at 565 (M-H)? in the ESIMS, and the molecular formula, C30H46O8S, was established by HRESIMS [565.2835, (M-H)?, 4?C group by Students mock cells by Dunnetts multiple Mrp2, where the concentrations of 2 and 1 in the bile of SD rats injected enterohepatic circulation. This was supported by the biphasic profile of plasma GA concentration in SD rats orally Toloxatone treated with GA. Finally, GA would be eliminated into the feces as GA or 3. On the other hand, the concentrations of 3MGA in the plasma and the urine of EHBRs orally treated with GA were much lower than those of 1 1, 2, and 3, exposing that 3MGA is usually a minor metabolite of GA in EHBRs. We found that most of 2 and 3 in plasma was bound to albumin, suggesting that these two compounds would not be eliminated into the urine glomerular filtration, but rather tubular secretion. As 1 experienced a high binding ratio to albumin and was a substrate for OAT1 and 318 and 2 and 3 share a high binding ratio to albumin, these compounds can be transported into the cells actively OAT1 and 3, and eliminated into the urine by tubular secretion. As GA also has a high binding ratio to albumin but is not a substrate for OATs, GA was not detected Tmem47 in the urine in either SD rats or EHBRs. The IC50 values of GL and its metabolites on 11because 111.0, MeOH); UV (MeOH) 6289) nm; ECD (MeOH) (565 [M-H]?; HRESIMS 565.2835 [M-H]? (calcd for C30H45O8S, 565.2835). 18(5.58 (1H, s, H-12), 3.95 (1H, dd 12.0, 4.5?Hz, H-3), 2.73 (1H, brd 13.5?Hz, H-1a), 2.48 (1H, s, H-9), 2.20 (1H, brd 14.5?Hz, H-18), 2.14 (1H, dd 14.0, 4.5?Hz, H-16a), 2.07 (1H, m, H-2a), 1.94 (1H, m, H-21a), 1.87 (1H, m, H-15a), 1.85 (1H, m, H-19a), 1.81 (1H, m, H-2b), 1.75 (1H, m, H-7a), 1.72 (1H, m, H-19b), 1.65 (1H,brd 13.0?Hz, H-6a), 1.60 (1H, m, H-6b), 1.45 (1H, m, H-7b), 1.44 (3H, s, H-27), 1.41 (1H, m, H-21b), 1.40 (2H, m, H-22), 1.25 (1H, brd 12.5?Hz, H-15b), 1.17 (3H, s, H-29), 1.16 (3H, s, H-25), 1.15 (3H, s, H-26), 1.06 (3H, s, H-23), 1.05 (1H, m, H-1b), 1.04 (1H, m, H-16b), 0.87 (1H, m, H-5), 0.86 (3H, s, H-24), 0.84 (3H, s, H-28) and 13C-NMR (CD3OD, 125?MHz) 202.6 (C-11), 180.5 (C-30), 172.9 (C-13), 128.9 (H-12), 87.2 (C-3), 63.0 (C-9), 56.6 (C-5), 49.9 (C-18), 46.7 (C-8), 44.9 (C-20), 44.6 (C-14), 42.4 (C-19), 40.1 (C-1), 39.9 (C-4), 39.0 (C-22), 38.2 (C-10), 33.8 (C-7), 33.0 (C-17), 32.0 (C-21), 29.2 (C-28), 28.7 (C-23), 28.7 (C-29), 27.6 (C-15), 27.4 (C-16), 25.2 (C-2), 23.8 (C-27), 19.3 (C-26), 18.6 (C-6), 17.0 (C-25), 16.9 (C-24); ESIMS 549 [M-H]-; HRESIMS 549.2888 [M-H]- (calcd for C30H45O7S, 549.2886). Determination of 11for 7?min), the concentrations of compounds 1, 2, 3, 3MGA, GA, and GL in the supernatant of the samples prepared from plasma and urine were measured using LC-ESIMS/MS under the following conditions: column, Scherzo SM-C18 (3?m, 3?mm for compound 1 (5.5?min), ESI(?) 565.5 to 96.5?for 2 (6.2?min), ESI(?) 549.5 to 96.5?for 3 (8.1?min), ESI(+) 647.6 to 453.6?for 3MGA (9.2?min), ESI(+) 471.3 to 91.0?for GA (8.8?min), ESI(+) 823.5 to 453.6?for GL (10.8?min), and ESI(+) 785.4 to 143.0?for astragaloside IV (3.0?min). Linear regressions over the concentration range of 32?nM to 20?M for each compound were examined using the peak-area ratio of the compounds to their internal requirements and the least-squares method ( em r /em 2? ?0.98). Uptake of compounds 2 and 3 by rat kidney slices and cells expressing OAT1 and 3 Uptake studies using rat kidney slices and cells stably expressing OAT1 and OAT3 were conducted with the same protocol as in our previous study18. We used the same pooled plasma of female EHBRs orally treated with GA for 12?h as in our previous study18. Assay of binding of compounds 2 and 3 to serum albumin We measured the binding ratios of compounds 2 and 3 to serum albumin by the same protocol using pooled plasma of female EHBRs as in our previous study18. Patient suffering from licorice-induced pseudoaldosteronism The patient was a 76-year-old female. She provided written informed consent to the use of.Therefore, it is predicted that there are other metabolites of GA that are biosynthesized by one- or two-step metabolic reactions among these three-step reactions. In the present study, we isolated two additional metabolites of GL from the urine of EHBRs orally treated with GA and identified their chemical structures. treated 1.0, MeOH)] exhibited a deprotonated molecule at 565 (M-H)? in the ESIMS, and the molecular formula, C30H46O8S, was established by HRESIMS [565.2835, (M-H)?, 4?C group by Students mock cells by Dunnetts multiple Mrp2, where the concentrations of 2 and 1 in the bile of SD rats injected enterohepatic circulation. This was supported by the biphasic profile of plasma GA concentration in SD rats orally treated with GA. Finally, GA would be eliminated into the feces as GA or 3. On the other hand, the concentrations of 3MGA in the plasma and the urine of EHBRs orally treated with GA were much lower than those of 1 1, 2, and 3, revealing that 3MGA is a minor metabolite of GA in EHBRs. We found that most of 2 and 3 in plasma was bound to albumin, suggesting that these two compounds would not be eliminated into the urine glomerular filtration, but rather tubular secretion. As 1 had a high binding ratio to albumin and was a substrate for OAT1 and 318 and 2 and 3 share a high binding ratio to albumin, these compounds can be transported into the cells actively OAT1 and 3, and eliminated into the urine by tubular secretion. As GA also has a high binding ratio to albumin but is not a substrate for OATs, GA was not detected in the urine in either SD rats or EHBRs. The IC50 values of GL and its metabolites on 11because 111.0, MeOH); UV (MeOH) 6289) nm; ECD (MeOH) (565 [M-H]?; HRESIMS 565.2835 [M-H]? (calcd for C30H45O8S, 565.2835). 18(5.58 (1H, s, H-12), 3.95 (1H, dd 12.0, 4.5?Hz, H-3), 2.73 (1H, brd 13.5?Hz, H-1a), 2.48 (1H, s, H-9), 2.20 (1H, brd 14.5?Hz, H-18), 2.14 (1H, dd 14.0, 4.5?Hz, H-16a), 2.07 (1H, m, H-2a), 1.94 (1H, m, H-21a), 1.87 (1H, m, H-15a), 1.85 (1H, m, H-19a), 1.81 (1H, m, H-2b), 1.75 (1H, m, H-7a), 1.72 (1H, m, H-19b), 1.65 (1H,brd 13.0?Hz, H-6a), 1.60 (1H, m, H-6b), 1.45 (1H, m, H-7b), 1.44 (3H, s, H-27), 1.41 (1H, m, H-21b), 1.40 (2H, m, H-22), 1.25 (1H, brd 12.5?Hz, H-15b), 1.17 (3H, s, H-29), 1.16 (3H, s, H-25), 1.15 (3H, s, H-26), 1.06 (3H, s, H-23), 1.05 (1H, m, H-1b), 1.04 (1H, m, H-16b), 0.87 (1H, m, H-5), 0.86 (3H, s, H-24), 0.84 (3H, s, H-28) and 13C-NMR (CD3OD, 125?MHz) 202.6 (C-11), 180.5 (C-30), 172.9 (C-13), 128.9 (H-12), 87.2 (C-3), 63.0 (C-9), 56.6 (C-5), 49.9 (C-18), 46.7 (C-8), 44.9 (C-20), 44.6 (C-14), 42.4 (C-19), 40.1 (C-1), 39.9 (C-4), 39.0 (C-22), 38.2 (C-10), 33.8 (C-7), 33.0 (C-17), 32.0 (C-21), 29.2 (C-28), 28.7 (C-23), 28.7 (C-29), 27.6 (C-15), 27.4 (C-16), 25.2 (C-2), 23.8 (C-27), 19.3 (C-26), 18.6 (C-6), 17.0 (C-25), 16.9 (C-24); ESIMS 549 [M-H]-; HRESIMS 549.2888 [M-H]- (calcd for C30H45O7S, 549.2886). Determination of 11for 7?min), the concentrations of compounds 1, 2, 3, 3MGA, GA, and GL in the supernatant of the samples prepared from plasma and urine were measured using LC-ESIMS/MS under the following conditions: column, Scherzo SM-C18 (3?m, 3?mm for compound 1 (5.5?min), ESI(?) 565.5 to 96.5?for 2 (6.2?min), ESI(?) 549.5 to 96.5?for 3 (8.1?min), ESI(+) 647.6 to 453.6?for 3MGA (9.2?min), ESI(+) 471.3 to 91.0?for GA (8.8?min), ESI(+) 823.5 to 453.6?for GL (10.8?min), and ESI(+) 785.4 to 143.0?for astragaloside IV (3.0?min). Linear regressions over the concentration range of 32?nM to 20?M for each compound were examined using the peak-area ratio of the compounds to their internal standards and the least-squares method ( em r /em 2? ?0.98). Uptake of compounds 2 and 3 by rat kidney slices and cells expressing OAT1 and 3 Uptake studies using rat kidney slices and cells stably expressing OAT1 and OAT3 were conducted with the same protocol as in our previous study18. We used the same pooled plasma of female EHBRs orally.O.M. rhabdomyolysis. Results Isolation and structural elucidation of compounds 2 and 3 from EHBR urine Female EHBRs were administered 1?mg/ml GA drinking water for 3 months, and their urine was collected and pooled. From 1 liter of this urine, we isolated 2 (1.9?mg) as a new GA metabolite, and 3 (0.4?mg) that was first isolated from the bile of rats treated 1.0, MeOH)] exhibited a deprotonated molecule at 565 (M-H)? in the ESIMS, and the molecular formula, C30H46O8S, was established by HRESIMS [565.2835, (M-H)?, 4?C group by Students mock cells by Dunnetts multiple Mrp2, where the concentrations of 2 and 1 in the bile of SD rats injected enterohepatic circulation. This was supported by the biphasic profile of plasma GA concentration in SD rats orally treated with GA. Finally, GA would be eliminated into the feces as GA or 3. On the other hand, the concentrations of 3MGA in the plasma and the urine of EHBRs orally treated with GA were much lower than those of 1 1, 2, and 3, revealing that 3MGA is a minor metabolite of GA in EHBRs. We found that most of 2 and 3 in plasma was bound to albumin, suggesting that these two compounds would not be eliminated into the urine glomerular filtration, but rather tubular secretion. As 1 had a high binding ratio to albumin and was a substrate for OAT1 and 318 and 2 and 3 share a high binding ratio to albumin, these compounds can be transported into the cells actively OAT1 and 3, and eliminated into the urine by tubular secretion. As GA also has a high binding ratio to albumin but is not a substrate for OATs, GA was not recognized in the urine in either SD rats or EHBRs. The IC50 ideals of GL and its metabolites on 11because 111.0, MeOH); UV (MeOH) 6289) nm; ECD (MeOH) (565 [M-H]?; HRESIMS 565.2835 [M-H]? (calcd for C30H45O8S, 565.2835). 18(5.58 (1H, s, H-12), 3.95 (1H, dd 12.0, 4.5?Hz, H-3), 2.73 (1H, brd 13.5?Hz, H-1a), 2.48 (1H, s, H-9), 2.20 (1H, brd 14.5?Hz, H-18), 2.14 (1H, dd 14.0, 4.5?Hz, H-16a), 2.07 (1H, m, H-2a), 1.94 (1H, m, H-21a), 1.87 (1H, m, H-15a), 1.85 (1H, m, H-19a), 1.81 (1H, m, H-2b), 1.75 (1H, m, H-7a), 1.72 (1H, m, H-19b), 1.65 (1H,brd 13.0?Hz, H-6a), 1.60 (1H, m, H-6b), 1.45 (1H, m, H-7b), 1.44 (3H, s, H-27), 1.41 (1H, m, H-21b), 1.40 (2H, m, H-22), 1.25 (1H, brd 12.5?Hz, H-15b), 1.17 (3H, s, H-29), 1.16 (3H, s, H-25), 1.15 (3H, s, H-26), 1.06 (3H, s, H-23), 1.05 (1H, m, H-1b), 1.04 (1H, m, H-16b), 0.87 (1H, m, H-5), 0.86 (3H, s, H-24), 0.84 (3H, s, H-28) and 13C-NMR (CD3OD, 125?MHz) 202.6 (C-11), 180.5 (C-30), 172.9 (C-13), 128.9 (H-12), 87.2 (C-3), 63.0 (C-9), 56.6 (C-5), 49.9 (C-18), 46.7 (C-8), 44.9 (C-20), 44.6 (C-14), 42.4 (C-19), 40.1 (C-1), 39.9 (C-4), 39.0 (C-22), 38.2 (C-10), 33.8 (C-7), 33.0 (C-17), 32.0 (C-21), 29.2 (C-28), 28.7 (C-23), 28.7 (C-29), 27.6 (C-15), 27.4 (C-16), 25.2 (C-2), 23.8 (C-27), 19.3 (C-26), 18.6 (C-6), 17.0 (C-25), 16.9 (C-24); ESIMS 549 [M-H]-; HRESIMS 549.2888 [M-H]- (calcd for C30H45O7S, 549.2886). Dedication of 11for 7?min), the concentrations of compounds 1, 2, 3, 3MGA, GA, and GL in the supernatant of the samples prepared from plasma and urine were measured using LC-ESIMS/MS under the following conditions: column, Scherzo SM-C18 (3?m, 3?mm for compound 1 (5.5?min), ESI(?) 565.5 to 96.5?for 2 (6.2?min), ESI(?) 549.5 to 96.5?for 3 (8.1?min), ESI(+) 647.6 to 453.6?for 3MGA (9.2?min), ESI(+) 471.3 to 91.0?for GA (8.8?min), ESI(+) 823.5 to 453.6?for GL (10.8?min), and ESI(+) 785.4 to 143.0?for astragaloside IV (3.0?min). Linear regressions on the concentration range of 32?nM to 20?M for each compound were examined using the peak-area percentage of the compounds to their internal requirements and the least-squares method.