ITSP Extraction of Cortisol from Plasma

Transcription

1 ITSP Extraction of Cortisol from Plasma Introduction Solid Phase Extraction (SPE) is well established as a preferred tool for extracting small organic molecules from complex biological matrices (e.g. plasma) because of efficient matrix removal. This paper details use of the ITSP SPE device and LC/MS to successfully quantify cortisol levels in rat plasma. Background For decades SPE cartridges have been used to enrich or extract target molecules from complex matrices. Because of the efficiency in sample preparation, SPE is the gold standard method for many sample preparation procedures. However, traditional SPE cartridge and plate implementation suffers from difficult automation and extensive method development requirements. So, while SPE is generally regarded as the best method of sample prep, it is often the last choice because of the extensive labor requirements. To bypass the labor requirements of traditional SPE, on-line concentration and extraction approaches have gained popularity in recent years. These on-line trap and elute systems are fully automated and have demonstrated efficient performance for many traditional SPE applications. However, these on-line approaches suffer many shortcomings: Complex hardware and software setup. Expensive up-front costs for equipment acquisition and setup. Extensive effort to validate trap column regeneration between samples Potential carryover risk with the trapping column The ITSP format was developed to overcome these shortcomings by facilitating automated sample prep (SPE, filtration) using common laboratory robots (e.g. CTC PAL). pans wanted to evaluate the ITSP claims and see if setting up an ITSP SPE system really was as easy and effective as it seemed. A common SPE application, the extraction of steroids from plasma, was chosen as the test application. Two common steroids, cortisol and cortisone were chosen as the analytes of interest. H H H H H Cortisol C21H305 MW=362.5 Cortisone C21H285 MW=360.5

2 Apparatus A Leap PAL was used to prepare the samples while an Agilent LC/MS system was used to analyze the samples. The instruments were configured as follows: Leap PAL: A standard LC PAL with a 100 L L-Mark syringe and two trays. Each tray holds 2 microtiter plates. The ITSP hardware kit from Microliter. Agilent LC/MS: A 1200 Rapid Resolution HPLC stack including solvent degasser, binary high pressure pump, wellplate autosampler, and column oven. The MS was a 1956B single quad with the multimode source. ITSP cartridges: 10mg rochem C18 LC-MS Conditions Solvent A was water with 0.05% TFA and solvent B was methanol with 0.05% TFA. Five microliters of the sample was injected on a Zorbax XDB C m 3.0x30mm column. The flow rate was 1.5 ml/min and the column temperature was 55 o C. The following solvent gradient was used to separate and elute the analytes. Time %B MS ionization was by simultaneous multi-mode (+APCI+ESI). Cortisone was detected by selected ion monitoring of m/z=361 while cortisol was detected at m/z=363. Cortisone was used as an internal standard while cortisol was the quantified analyte. Standards were prepared in 50/50 Water/Methanol with a constant cortisone concentration of 100 ng/ml and cortisol concentrations from 3 to 2500 ng/ml. The standards were analyzed using the LC-MS conditions above to produce the following calibration plots.

3 Sample Prep: In order to test the ITSP SPE device, two sets of samples were created covering a cortisol concentration range of 31 to 2500 ng/ml with a constant cortisone concentration of 100 ng/ml. The solvent set was in 90/10 Water/Methanol and the plasma set was spiked into rat plasma. Various conditions were evaluated and the results from these are discussed in the bservations section. The final extraction protocol was as follows: 1. The ITSP SPE cartridge was washed with 100 L of Acetonitrile at 10 L/sec flow. 2. The ITSP was washed with 100 L of Water (0.2% Formic Acid) at 10 L/sec flow L of sample was loaded onto the ITSP at 5 L/sec flow. 4. The ITSP cartridge was moved over collection vial 1 and washed with 30 L of 20% Acetonitrile in water (0.2% FA) at 5 L/sec. 5. The ITSP cartridge was moved over collection vial 2 and eluted with 80 L of 80% Acetonitrile in water (0.2% FA) at 5 L/sec. 6. The ITSP cartridge was moved over collection vial 3 and eluted with 80 L of 100% Acetonitrile (0.2% FA) at 5 L/sec. 7. The vials were capped. 8. The vials were vortexed to insure complete mixing. 9. The vials were placed in the LC-MS autosampler. 10. The samples were analyzed using the LC-MS method. 11. Peak areas were determined using Chemstation software. 12. The following results were obtained for the second elution fraction (vial 2)

4 Discussion In all respects, the ITSP SPE device performed as expected. The linearity and precision were both very good. The absolute recovery for standards in solvent was excellent averaging 100% across the calibration range. The recovery from plasma was acceptable at 80%. In general, extraction of cortisone from plasma was more efficient than extraction of cortisol. In data not reported, recoveries near 100% were observed for cortisone when

5 eluting with 80% acetonitrile in water while they were 60% for cortisol. The addition of formic acid to the elution solvent significantly improved the recovery for cortisol. For both plasma and solvent standards processed by ITSP, better than 95% of the sample was recovered in the single elution fraction #2. The initial wash fraction of 20% acetonitrile and the final elution fraction of 100% acetonitrile contained very little cortisone or cortisol. While these other two fractions were analyzed for verification of performance, the results are not reported. Various sample volumes were investigated. The 10 mg cartridges tested were well suited to injection volumes between 10 and 60 microliters. This volume range matches nicely with typical sample volumes from in-vivo studies. Forty microliters was chosen because it maximized sensitivity without risk of cartridge overload. The cartridges have a low retained volume of only a few microliters. The exact volume depends on the properties of the solvent; with the cartridge retaining more water than acetonitrile. ptimum wash and elution volumes range from 30 to 100 microliters. Recovery may decrease with lower elution volumes while sample blowthrough may be observed with extensive washing. Elution volumes greater than 100 microliters only seem to dilute the sample with a negligible increase in total recovery. The result of a 40 L load on the ITSP and an 80 L elution was a minimal factor of 2X sample dilution. Typical SPE systems and/or acetonitrile protein precipitation protocols result in sample dilutions between 4 and 10X. The advantage of the ITSP is increased sensitivity without the evaporation/reconstitution step often used. As a general rule, ITSP injection flow rates are best kept below 10 L/s. Higher flow rates are possible with low viscosity organic solvents. Also, a protocol with few septum punctures will maintain the septum integrity thereby supporting positive pressure solvent delivery. We did not observe any problems with 10 L/s conditioning and 5 L/s elution steps. Perhaps the greatest advantage of the ITSP over other SPE formats is the ease of automated mobility. The standard configuration PAL autosampler easily and reliably moved the ITSP device from the prep plate to different elution locations. The ability to elute from the same device into an infinite number of locations greatly increases the automation potential for method development. Conclusions The ITSP is a solid format for building high quality automated SPE methods. The device is well suited for PAL automation. The sample and elution volumes are optimum for typical preparation of in-vivo samples for HPLC/MS analysis.