Effect of Analyte Desorption on Sensitivity in Dried Blood Spot Analysis
Ritu Arora, William C. Hudson, Paul Boguszewski , Ben Yong, and William Long
Agilent Technologies, 25200 Commercentre Drive, Lake Forest, CA 92630
EAS 2011Nov 16 - Wed Poster
Results and Discussion
Results and Discussion
b) Acidic analytes – Statins
Column: Poroshell 120 EC-C8, 2.7µ, 150 x 2.1 mm
(Part #: 693775-906)
Mobile Phase: A: 5 mM Ammonium formate, B: ACN
Flow rate: 200 µL/ min
Gradient: t0 A: 70%, B: 30%
t5.0 A: 25%, B: 75%
t5.5 A: 25%, B: 75%
t5.6 A: 70%, B: 30%
t8.0 A: 70%, B: 30%
Colum Temp.: 30°C
Run Time: 8:00 min
Gas Temp.: 275°C Gas Flow: 10 L/min
Nebulizer: 10 psi
Sheath Gas Temp.: 250°C Sheath Gas Flow: 7 L/min
CompoundQ1 ionProduct ionCE (V)
Atenolol 267.2 145.1 22
Pindolol 249.2 116.1 14
Metoprolol 268.2 116.2 14
Propranolol 260.2 116.1 14
Atorvastatin 557.2 397.1 27
Simvastatin 435.3 318.9 11
Pravastatin 423.2 321.1 7
Lovastatin 421.3 318.9 11
Results and Discussion
A) Desorption methods
Dried blood spot (DBS) technology combined with the analytical capability of modern mass spectrometers (LC-MS/MS) has recently emerged as an important method in the quantitative bioanalysis of small molecules. Its great interest lies in the small volume of sample required, ease of collection, reduced sample shipping costs, and versatile storage conditions.
As a relatively new technique in bioanalysis, it is essential to investigate the impact of variables that may affect the overall efficiency of the technique. A novel, non-cellulose based dried blood spotting material has been evaluated. This work focuses on method development for optimal desorption methods and desorption solvents. Their effects on analyte desorption and resulting sensitivity were investigated.
- B) Centrifugation time
- The centrifugation time was increased in 15 min increments to test if recoveries/responses could be improved with higher centrifuge times. 3 mm spots were taken from different 20 ng/mL spots and put into 2 mL centrifuge tubes. 0.1% formic acid 80:20 MeOH:H2O was added to each spot and centrifuged for 15, 30, 45, and 60 minutes. Each sample was then evaporated and reconstituted in 100 µL of mobile phase.
- C) Desorption solvents
- Both MeOH and ACN were tested at various concentrations with 0.1% formic acid, i.e. 100%, 80%, 60%, and 40% organic. A concentration study of formic acid was also carried out with 80% MeOH and ACN - 0%, 0.1%, 0.5% and 1% formic acid. 3 mm spot were taken from different 20 ng/mL spots and put into 2 mL centrifuge tubes. They were then desorbed with 300 µL of each of the desorption solvents, centrifuged at 15,000 rpm for 15 minutes, evaporated, and reconstituted in 100 µL mobile phase.
- Acidic analytes
- A) Desorption solvents
- Two sets of experiments were conducted to determine the optimal desorption solvent needed for achieving best sensitivity for statins overall. Blood spiked with statins mix at 20 ng/ml was spotted on DMS cards, and dried overnight. 3 mm spots were punched, and each punch was dissolved in 300 µL of desorption solvent, left to soak for ~ 2 hours, followed by evaporation to dryness, and final reconstitution in 100 µL of mobile phase for LC/MS analysis.
- Desorption solvents investigated included neat acetonitrile and methanol, different concentrations of both organic solvents (100% and 80%) with and without modifiers such as ammonium formate and ammonium hydroxide. Modifiers were added to promote ionization of acidic compounds, such as statins.
- After seeing which of the above resulted in best sensitivity, further experiments were carried out with 80% MeOH with 1% NH4OH as the reference, and MeOH % varied (80%, 60%, and 40%).
- B) Desorption methods
- Centrifugation (15 min) vs. soak (one hour) - 3 mm DMS blood spots were desorbed using 300 µL 60% MeOH with 1% NH4OH, samples were centrifuged at 15000 rpm for 15 minutes, evaporated to dryness, and reconstituted in 100 µL mobile phase. Another set of spots were desorbed in the same solvent, and soaked for one hour before evaporation.
- LC-MS Conditions - Agilent 1290 LC / 6460 QQQ
- a) Basic analytes - β-blockers
- Column: Poroshell 120 EC-C18, 2.7 µm, 30 x 2.1 mm
- Mobile Phase: A: 0.1% Formic Acid in H2O, B: MeOH
- Flow rate: 200 µL/ min
- Gradient: t0 A: 80%, B: 20%
- t1.0-2.0A: 20%, B: 80%
- t2.01-3.0 A: 80%, B: 20%
- Run Time: 3:00 min
- Gas Temp: 325°C, Gas Flow: 10 L/min
- Nebulizer: 15 psi
- Sheath Gas Temp: 250°C, Sheath Gas Flow: 7 L/min
- Polarity: Positive
B) Centrifugation time
MeOH clearly worked better than ACN for the more hydrophilic pravastatin. NH4OH worked better than ammonium formate buffer.
100% ACN / 0.1% FA generates the best response. In general, ACN works better than MeOH for every organic % tried. In the FA concentration study, the best response was given with 0.5% FA in 80% ACN.
Table 1. Basic drugs screened - general information
- Centrifugation for 15 min time appears to be enough. Propranolol decreased with greater time, probably due to more interferences being desorbed with more time.
- C) Desorption solvents
Compound Log P pKa Therapeutic use
Atenolol 0.5 9.6 β-blocker, antihypertensive
Pindolol 1.9 8.8 β-blocker
Metoprolol 1.6 9.7 β-blocker, antihypertensive
Propranolol 3.0 9.5 β-blocker, antihypertensive
Table 2. Acidic drugs (statins) screened - general information
- For the more hydrophobic propranolol, MeOH works better than ACN in general, with 80% MeOH / 0.1% FA yielding the best response. In the FA concentration study, 0.1% FA in 80% MeOH is far above the other concentrations.
- Acidic analytes
- A) Desorption solvents - Atorvastatin
60% CH3OH with 1% NH4OH appeared to be the optimal desorption solvent for this group of compounds as both simvastatin and lovastatin yielded best response, atorvastatin being extremely sensitive resulted in a much higher response than the other analytes even though this was not the best solvent for it, and pravastatin gave a measurable response. Pravastatin, being the most hydrophilic of all, gave best results with the highest aqueous solvent, 40% CH3OH. Nevertheless, it was not chosen as higher aqueous desorption solvents would result in dirtier extracts.
B) Desorption methods – Centrifugation vs. Soak - Atorvastatin
- Basic analytes
- Fresh human whole blood was spiked with a mix of four basic pharmaceuticals, comprising β-blockers such as atenolol, pindolol, metoprolol, and propranolol, at a concentration of 20 ng/mL. After vortexing, 15µL of blood was aliquoted per spot on Agilent Bond Elut Dried Matrix Spotting (DMS) cards, followed by overnight drying. Circular punches of 3 mm in diameter were taken from the DMS cards and subjected to the following experiments:
- A) Desorption methods
- Different techniques were tested to evaluate the best way to desorb the analytes from the membrane. Each test was compared to a standard and protein precipitation, All blood samples were evaporated and reconstituted in 100 µL of mobile phase.
- Standard - 4 µL of 20 ng/mL standard was desorbed with 300 µL 0.1% formic acid 80:20 MeOH:H2O, centrifuged at 15,000 rpm for 15 mins, evaporated to dryness, and diluted to 100 µL mobile phase.
- Protein precipitation - 4 µL of blood, was diluted to 100 µL with H2O. 300 µL of 0.1% formic acid in MeOH was used as a crash solvent (1:3 aqueous:organic crash).
- Centrifugation (15 min) - a 3 mm DMS blood spot was desorbed using 300 µL 0.1% formic acid 80:20 MeOH:H2O. The sample was centrifuged at 15000 rpm for 15 minutes.
- Soak (one hour) - a 3 mm DMS blood spot was desorbed using 300 µL 0.1% formic acid 80:20 MeOH:H2O. The sample was soaked for 1 hour before evaporation.
Atenolol yields the best response at 60% ACN / 0.1% FA or 40% MeOH / 0.1% FA. In the FA concentration study experiments, the best response was given with 0.5% FA in 80% ACN.
Centrifugation yields a somewhat better response compared to soak for 60 mins for this analyte.
Since atorvastatin was the most sensitive of all statins, looked at average response on this analyte as opposed to the other statins. MeOH worked significantly better when compared to ACN for most of the 100% experiments, most likely due to a solubility issue. 80% MeOH neat and with modifiers yielded improved responses compared to their ACN counterparts. NH4OH worked better than ammonium formate buffer.
- Analyte desorption and its resulting impact on sensitivity is better when centrifugation is adopted in comparison to soak for one hour for both basic and acidic analytes investigated.
- A 15 min centrifugation time appears to be appropriate and longer centrifugation times do not offer increased sensitivity.
- Desorption solvents for basic compounds like β-blockers: There is no generic method for all compounds. Polar compounds like atenolol, pindolol, and metoprolol work better with ACN, while nonpolaranalytes like propranolol yield better responses with MeOH. Pindolol works best with no FA but propranolol looks very poor. The best overall method appears to be 80% MeOH with 0.1% FA. It is the best solvent for propranolol and the other compounds are good enough.
- Optimal desorption conditions for acidic analytes such as statins: The solvent which yielded the best sensitivity overall was 60% CH3OH with 1% NH4OH.
Analyte desorption was measured in terms of MS response for each analyte. The effect of different desorption methods, centrifugation times, and desorption solvents on analyte sensitivity is presented, thereby reflecting on the overall efficiency of the technique.
Centrifuging the sample for 15 minutes gives the best overall results, especially for the more hydrophobic propranolol. Atenolol which is the most polar has the poorest recovery.
60% ACN / 0.1% FA generates the best response. No FA in 80% ACN or MeOH worked best.