Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Synthesis of Salbutamol-4′-O-sulfate as Reference
2.3. Characterization of Salbutamol-4′-O-sulfate
2.3.1. UHPLC-QTOF-MS
2.3.2. Nuclear Magnetic Resonance
2.4. Proof of Concept: Longitudinal Case Study and Urine Analysis
2.4.1. Study Design
2.4.2. Matrix Assisted Calibration
2.4.3. Sample Preparation
2.4.4. Specific Gravity of Urine Samples
2.4.5. Instruments and Chromatographic Conditions for Urine Analysis
2.4.6. Method Characterization
2.5. Data Analysis
3. Results
3.1. Biosynthesis and Characterization of Reference by UHPLC-QTOF-MS and NMR
3.2. Metabolite Identification in Urine Samples
3.3. UHPLC-QQQ-MS/MS Method Characterization
3.4. Evaluation of Urinary Excretion Profiles
3.4.1. Inhalation of Salbutamol through Dry Powder Inhaler vs. Metered Dose Inhaler
3.4.2. Urinary Excretion Rates
3.4.3. Proportions of Salbutamol and Salbutamol-4′-O-sulfate
3.4.4. Salbutamol-4′-O-sulfate in Relation to Unchanged Salbutamol
3.4.5. Adjustment by the Specific Gravity of the Urine and by Urinary Flow Rate
4. Discussion
4.1. Biosynthesis and Characterization of Salbutamol-4′-O-sulfate
4.2. Basic Method Validation
4.3. Proof of Concept: Achiral Analysis of Urinary Excreted Salbutamol and Salbutamol-4′-O-sulfate for Discrimination of Application Routes and Enantiomeric Composition of the Administered Drug
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Appendix A.1. Purification of Biosynthesized Salbutamol-4′-O-sulfate
Gravity Column Purification | HPLC Purification | |
---|---|---|
Sample preparation | Dissolving dried remains in methanol, filtration | Evaporation of fractions containing product to reduce sample volume Filtration of silica remains |
Stationary phase | Silica | C18 |
Column length | 40 cm | 25 cm |
Column diameter | 3.5 cm | 1 cm |
Particle size | n.a. | 5 µm |
Flow rate | n.a. | 2.5 mL/min |
Mobile phase | Isopropanol:ethyl acetate:ammonia (17.5%) 40:50:10 (V:V:V) | A: water B: acetonitrile 0–5 min: 3% B to 15% B 5–6.5 min: 15% B 6.5–16 min: 15% B to 27% B 16–20 min: 27% B to 45% B 20–22 min: 45% B to 95% B 25–27 min: 95% B to 3% B |
Detection of product | Fraction analysis with LC-MS | UV detection 265 nm |
Appendix A.2. Inhalation of Salbutamol through Dry Powder Inhaler vs. Metered Dose Inhaler
DPI 1 | MDI 2 | MDI 2_2 | |
---|---|---|---|
Percentage of dose recovered in urine 3 | 80% | 115% | 83% |
Proportion of parent compound 4 | 42% | 46% | 41% |
Proportion of salbutamol-4′-O-sulfate 4 | 58% | 54% | 59% |
Appendix A.3. Appendix Figures
References
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Electrospray Ionization | ||||
---|---|---|---|---|
Gas temperature | 170 °C | |||
Gas flow | 17 L/min | |||
Nebulizer | 10 psi | |||
Sheath gas temperature | 400 °C | |||
Sheath gas flow | 12 L/min | |||
Capillary voltage | 4000 V | |||
Nozzle voltage | 500 V | |||
MRM | ||||
Precursor Ion [m/z] | Product Ion [m/z] | Collision Energy [eV] | ||
Salbutamol | [M+H]+ = 240.0 | 222.1 | 8 | |
166.1 | 12 | |||
148.1 * | 16 | |||
121.1 | 25 | |||
91.0 | 48 | |||
77.1 | 56 | |||
Salbutamol-4′-O-sulfate | [M+H]+ = 320.0 | 240.0 * | 4 | |
222.0 | 16 | |||
166.0 | 16 | |||
148.0 | 32 | |||
77.0 | 80 | |||
[M−H]− = 318.0 1 | 238.0 | 25 | ||
d9-Salbutamol | [M+H]+ = 249.2 | 231.1 | 8 | |
166.1 | 12 | |||
148.1 | 16 | |||
121.1 | 25 | |||
Salbutamol glucuronide | [M+H]+ = 416.0 1 | 298.0 | 12 | |
240.0 | 18 | |||
224.0 | 29 | |||
222.0 | 20 | |||
148.0 | 20 | |||
[M−H]− = 414.0 1 | 396.0 | 18 | ||
220.0 | 25 | |||
146.0 | 25 |
Salbutamol (a) | Salbutamol-4′-O-sulfate (a) | Chemical Shift Differences ∆δ= δOsulfate − δOH | ||||
---|---|---|---|---|---|---|
Position | 1H | 13C | 1H | 13C | 1H | 13C |
1 | 4.72, dd (b), 10.0, 2.8 Hz | 69.71 | 4.77, dd (b), 10.2, 2.6 Hz | 69.69 | +0.05 | –0.02 |
2 | 2.74/2.83, AB d (b) 11.8, 10.0, 2.8 Hz | 49.14 | 2.69/2.81 (c) | 49.67 | –0.05/–0.02 | +0.53 |
4 | 53.89 | 54.68 (d) | +0.79 | |||
5 | 1.20, s | 26.12 | 1.23, s | 25.91 | +0.03 | –0.21 |
1′ | 132.93 | 137.46 | +4.53 | |||
2′ | 7.31, d, 2.3 Hz | 125.88 | 7.43, d, 2.1 Hz | 124.86 | +0.12 | –1.02 |
3′ | 128.10 | 134.57 | +6.47 | |||
3′-CH2 | 4.47, s | 58.24 | 4.55, s | 58.30 | +0.08 | +0.06 |
4′ | 153.43 | 149.59 | –3.84 | |||
5′ | 6.73, d, 8.3 Hz | 114.16 | 7.27. d, 8.3 Hz | 121.46 | +0.54 | +7.30 |
6′ | 7.07, dd, 8.3, 2.3 Hz | 124.96 | 7.20, dd 8.3, 2.1 Hz | 121.50 | +0.13 | –3.46 |
Product Ion (m/z) | 77.1 | 91.0 | 121.1 | 166.1 | 222.1 |
---|---|---|---|---|---|
salbutamol | 12.1–22.1 | 4.0–14.0 | 5.1–15.1 | 26.4–39.6 | 59.8–79.8 |
Product Ion (m/z) | 148.0 | 166.0 | 222.0 | ||
salbutamol-4′-O-sulfate | 44.8–64.8 | 15.2–25.2 | 29.9–44.9 |
Salbutamol | Salbutamol-4′-O-sulfate | |
---|---|---|
tmax (urine) | ||
Oral racemate 2 mg (SAP) | 3 h | 3 h |
Inhaled aerosol racemate 600 µg (SA_MDI_2) | 1.5 h | 1.5 h |
Oral levosalbutamol 1 mg (LSAP) | 3 h | 0.5 h |
Inhaled levosalbutamol 630 µg (LSA_MDI) | 0.5 h | 3 h |
Inhaled levosalbutamol 90 µg (LSA_MDI_TD) | 0.5 h | 3 h |
Total urinary excretion 1 | ||
Oral racemate 2 mg (SAP) 1 | 449 µg (22.5%) | 1030 µg (51.5%) |
Inhaled aerosol racemate 600 µg (SA_MDI_2) 1 | 203 µg (33.8%) | 298 µg (49.6%) |
Oral levosalbutamol 1 mg (LSAP) 1 | 65 µg (6.5%) | 847 µg (84.7%) |
Inhaled levosalbutamol 630 µg (LSA_MDI) 1 | 129 µg (20.5%) | 371 µg (58.9%) |
Inhaled levosalbutamol 90 µg (LSA_MDI_TD) 1 | 19 µg (21.1%) | 55 µg (61.1%) |
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Jendretzki, A.L.; Harps, L.C.; Sun, Y.; Bredendiek, F.; Bureik, M.; Girreser, U.; de la Torre, X.; Botrè, F.M.; Parr, M.K. Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS. Separations 2023, 10, 427. https://doi.org/10.3390/separations10080427
Jendretzki AL, Harps LC, Sun Y, Bredendiek F, Bureik M, Girreser U, de la Torre X, Botrè FM, Parr MK. Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS. Separations. 2023; 10(8):427. https://doi.org/10.3390/separations10080427
Chicago/Turabian StyleJendretzki, Annika Lisa, Lukas Corbinian Harps, Yanan Sun, Felix Bredendiek, Matthias Bureik, Ulrich Girreser, Xavier de la Torre, Francesco M. Botrè, and Maria Kristina Parr. 2023. "Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS" Separations 10, no. 8: 427. https://doi.org/10.3390/separations10080427