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 2011 Nov 16 - Wed Poster. Introduction. Experimental.
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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
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
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
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
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 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.