Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Equipment
2.2. Instrumentation
Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry
2.3. Study Design and Sampling of Urine and Serum
2.4. Determination of Urinary Concentrations
2.4.1. Sample Preparation
2.4.2. Matrix-Matched Calibration of Salbutamol, Formoterol, and Salbutamol-4′-O-Sulfate
2.4.3. Determination of the Specific Gravity of Urine
2.5. Determination of Serum Concentrations
2.5.1. Development of Sample Preparation Protocol
2.5.2. Final Study Sample Preparation Protocol
2.5.3. Matrix-Matched Calibration of Salbutamol, Formoterol, and Salbutamol-4′-O-Sulfate
2.6. Method Performance Characterization
2.6.1. Matrix Effect
2.6.2. Recovery
2.6.3. Limit of Quantitation and Limit of Detection
2.6.4. Carry Over
2.6.5. Retention Time Stability
3. Results
3.1. SPE Method Development
3.2. UHPLC-MS/MS Method Performance Characterization
3.2.1. Limit of Quantitation and Limit of Detection
3.2.2. Carry Over
3.2.3. Retention Time Stability
3.2.4. Performance of Hydrolysis in Urine Samples by β-Glucuronidase
3.2.5. Recovery
3.2.6. Matrix Effect
3.3. Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate
3.3.1. Serum Sample Analysis
3.3.2. Urine Sample Analysis
4. Discussion
4.1. UHPLC-MS/MS and Sample Preparation
4.2. Urine Analysis
4.3. Serum Analysis
4.4. Phase II Metabolites
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Step | HLB | HR-XC |
---|---|---|
Priming | 3 mL MeOH | 3 mL MeOH |
Conditioning | 3 mL H2O | 3 mL H2O |
Samples application (slowly) | 1.0 mL | 1.0 mL |
Washing | 1 mL water | 1 mL 0.1 M HCl in water, then 1 mL of MeOH |
Drying SPE for 10 min under vacuum | Do not let run dry | |
Elution into a conical vial | 1 mL MeOH | 5% NH3 in 1 mL MeOH |
Drying under a stream of nitrogen at 40 °C | ||
Reconstitution in the same conical vial | 50 μL 0.1% formic acid in water (0.1:100, v:v) | 50 μL 0.1% formic acid in water (0.1:100, v:v) |
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Analytes | Salbutamol and Formoterol | Formoterol | Salbutamol-4′-O-Sulfate | Salbutamol and Salbutamol-4′-O-Sulfate |
---|---|---|---|---|
Specimen | Urine | Serum | Urine | Serum |
Column | Poroshell 120 EC-C18 (2.1 mm I.D. × 50 mm; 1.9 μm) | Poroshell 120 phenyl-hexyl (3.0 mm I.D. × 100 mm; 1.9 μm) | ||
Flow rate | 0.3 mL/min | 0.4 mL/min | ||
Column temperature | 35 °C | 35 °C | ||
Injection volume | 1 μL (30 μL) | 15 μL | 2 μL | 5 μL |
Solvent A | 0.1% formic acid and 10 mM ammonium formate in water (1:0.63:1000, v:m:v) | 20 mM ammonium formate in water (1.26:1000, m:v) | ||
Solvent B | 0.1% formic acid and 10 mM ammonium formate in water/ACN (1:0.63:120:900, v:m:v:v) | 20 mM ammonium formate in methanol (1.26:1000, m:v) | ||
Gradient | 0 min: 0% B | 0 min: 5% B | 0 min: 5% B | |
2 min: 7.5% B | 2 min: 5% B | 1 min: 5% B | ||
5 min: 40% B | 5 min: 40% B | 5 min: 40% B | ||
6 min: 95% B | 6 min: 100% B | 6 min: 95% B | ||
7.4 min: 95% B | 7.4 min: 100% B | 7.90 min: 95% B | ||
7.5 min: 0% B | 7.5 min: 5% B | 8 min: 5% B | ||
Post time | 3 min | 2.5 min | ||
Ion source parameters | ||||
Gas temperature | 290 °C | 170 °C | ||
Gas flow | 20 L/min | 17 L/min | ||
Nebulizer | 25 psi | 10 psi | ||
Sheath gas temperature | 400 °C | 400 °C | ||
Sheath gas flow | 12 | 12 | ||
Capillary | 6000 V | 4000 V | ||
Nozzle voltage | 500 V | 500 V |
Analyte/ISTD | Precursor Ion (m/z) | Product Ion (m/z) | Collision Energy (V) |
---|---|---|---|
Formoterol | 345 | 327.1 | 12 |
345 | 149 | 20 | |
345 | 121.1 | 32 | |
345 | 91.1 | 76 | |
345 | 77.1 | 80 | |
Salbutamol | 240 | 222 | 8 |
240 | 166 | 12 | |
240 | 147.9 | 16 | |
240 | 91 | 48 | |
240 | 77.1 | 56 | |
Salbutamol-4′-O-sulfate | 320 | 240 | 4 |
320 | 222 | 16 | |
320 | 166 | 16 | |
320 | 148 | 32 | |
320 | 77 | 80 | |
d3-Salbutamol | 243.2 | 225.2 | 8 |
243.2 | 151 | 20 | |
d3-Testosterone-glucuronide | 468.3 | 109 | 40 |
468.3 | 97 | 32 | |
468.3 | 84.9 | 84.9 | |
d3-Testosterone | 292.2 | 109 | 20 |
292.2 | 97 | 20 |
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Harps, L.C.; Bizjak, D.A.; Girreser, U.; Zügel, M.; Steinacker, J.M.; Diel, P.; Parr, M.K. Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS. Separations 2023, 10, 368. https://doi.org/10.3390/separations10070368
Harps LC, Bizjak DA, Girreser U, Zügel M, Steinacker JM, Diel P, Parr MK. Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS. Separations. 2023; 10(7):368. https://doi.org/10.3390/separations10070368
Chicago/Turabian StyleHarps, Lukas C., Daniel A. Bizjak, Ulrich Girreser, Martina Zügel, Jürgen M. Steinacker, Patrick Diel, and Maria Kristina Parr. 2023. "Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS" Separations 10, no. 7: 368. https://doi.org/10.3390/separations10070368