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A SENSITIVE AND SELECTIVE MICROSCALE SPE ......A SENSITIVE AND SELECTIVE MICROSCALE SPE METHOD FOR...
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A SENSITIVE AND SELECTIVE MICROSCALE SPE METHOD FOR THE DETERMINATION OF F 2 a IN RAT PLASMA BY LC/MS/MS OVERVIEW - Previous solid phase extraction (SPE) methods for isoprostanes included using a C18 reversed-phase material with an elution solvent of ethyl ace- tate:heptane:methanol (5:4:1) that required evapo- ration and reconstitution before LC/MS/MS analy- sis. Additionally, using this method often resulted in recoveries of only 60%. We were asked to help a customer develop a method to run 10,000 to 30,000 samples per year with a better SPE method with higher recoveries. Because of the need for higher throughput, we developed a simplified method on an ultra-low elution volume 96-well SPE plate utilizing a mixed-mode material. Sample re- coveries were greater than 90%. Using the low elution volume plates, there is a concentration factor of at least 2. The calibration range was 0.1 to 100 pg/uL and was linear for both water and plasma samples. The detection limit was 0.1 pg/uL. INTRODUCTION – F 2 -iosprostanes are biomarkers for oxidative stress in vivo that are stable and distributed throughout the body. They are formed from nonenzymatic autoxi- dation of arachidonic acid in cell membranes (1). The extent of systematic damage due to oxidative stress within the body can be determined by measur- ing levels of these isoprostanes in body fluids such as plasma and urine (1). Our challenge was to develop a simple, robust and high-throughput SPE method followed by LC/MS/MS analysis for prostaglandin F 2a , or isoprostane. The challenge in this analysis was achieving both high throughput and low detection and quantitation limits (LOD/LOQ’s). We know that solid phase extraction (SPE) in a 96-well plate format (2, 3) can meet the demand for low LOD/LOQ’s using less sample volume. In this experiment, we chose to develop the method on a novel ultra-low elution volume 96-well SPE plate containing a mixed-mode material. Additionally, our method of chroma- tographic analysis was an LC/MS/MS system to increase our level of sensitivity. 1. Chu, K. O., Wang, C. C., Rogers, M. S., Pang, C.P. Anal. Biochem. 2003, 316, 111. 2. Zweigenbaum, T., Heing, K., Steinborner, S., Wachst, T., Henion, J., Anal. Chem.,1999, 71, 2294. 3. Show, W.Z., Jiang, X., Beato, B.D., Naidong, W., Rapid Commun. Mass Spectrom., 2001, 15, 466. STRUCTURES- SPE METHOD- Device: Oasis ® MAX μElution Plate Part Number: 186001829 Condition: 200 μL MeOH Equilibrate: 200 μL H 2 O Mix/Load: 350 μL Spiked H 2 O or rat plasma (0.1to 100 pg/μL)and 350 μL ISTD (10 pg/μL) in H 2 O Wash 1: 200 μL H2O with 2% NH 4 OH (Lock on acidic analytes and remove salts/polars) Wash 2: 200 μL MeOH (remove neutral and basic interferences) Elute: 50 μL ACN:IPA (40:60) with 5% FA Dilute: 125 μL H 2 O with 2% NH 4 OH (total volume of 175 μL results in a concentration factor of 2) Ziling Lu, Jeffrey Mazzeo, Claude Mallet, Diane Diehl Chemistry Applied Technology Group Waters Corporation, Milford, MA USA OH O H COOH OH O H COOH OH O ISTD: 8-Iso Prostaglandin E 2 FW: 352.5 MRM: 351.0 → 315.0 Analyte: Prostaglandin F 2a FW: 354.5 MRM 353.0 → 193.0
Transcript of A SENSITIVE AND SELECTIVE MICROSCALE SPE ......A SENSITIVE AND SELECTIVE MICROSCALE SPE METHOD FOR...
A SENSITIVE AND SELECTIVE MICROSCALE SPE METHOD FOR THE DETERMINATION OF F2a IN
RAT PLASMA BY LC/MS/MS
OVERVIEW - Previous solid phase extraction (SPE) methods for isoprostanes included using a C18 reversed-phase material with an elution solvent of ethyl ace-tate:heptane:methanol (5:4:1) that required evapo-ration and reconstitution before LC/MS/MS analy-sis. Additionally, using this method often resulted in recoveries of only 60%. We were asked to help a customer develop a method to run 10,000 to 30,000 samples per year with a better SPE method with higher recoveries. Because of the need for higher throughput, we developed a simplified method on an ultra-low elution volume 96-well SPE plate utilizing a mixed-mode material. Sample re-coveries were greater than 90%. Using the low elution volume plates, there is a concentration factor of at least 2. The calibration range was 0.1 to 100 pg/uL and was linear for both water and plasma samples. The detection limit was 0.1 pg/uL.
INTRODUCTION – F2-iosprostanes are biomarkers for oxidative stress in vivo that are stable and distributed throughout the body. They are formed from nonenzymatic autoxi-dation of arachidonic acid in cell membranes (1). The extent of systematic damage due to oxidative stress within the body can be determined by measur-ing levels of these isoprostanes in body fluids such as plasma and urine (1). Our challenge was to develop a simple, robust and high-throughput SPE method followed by LC/MS/MS analysis for prostaglandin F2a, or isoprostane.
The challenge in this analysis was achieving both high throughput and low detection and quantitation limits (LOD/LOQ’s). We know that solid phase extraction (SPE) in a 96-well plate format (2, 3) can meet the demand for low LOD/LOQ’s using less sample volume. In this experiment, we chose to develop the method on a novel ultra-low elution volume 96-well SPE plate containing a mixed-mode material. Additionally, our method of chroma-tographic analysis was an LC/MS/MS system to increase our level of sensitivity. 1. Chu, K. O., Wang, C. C., Rogers, M. S., Pang, C.P. Anal. Biochem. 2003, 316, 111. 2. Zweigenbaum, T., Heing, K., Steinborner, S., Wachst, T., Henion, J., Anal. Chem.,1999, 71, 2294. 3. Show, W.Z., Jiang, X., Beato, B.D., Naidong, W., Rapid Commun. Mass Spectrom., 2001, 15, 466.
STRUCTURES-
SPE METHOD- Device: Oasis® MAX µElution Plate Part Number: 186001829 Condition: 200 µL MeOH Equilibrate: 200 µL H2O Mix/Load: 350 µL Spiked H2O or rat plasma (0.1to 100 pg/µL)and 350 µL ISTD (10 pg/µL) in H2O Wash 1: 200 µL H2O with 2% NH4OH (Lock on acidic analytes and remove salts/polars) Wash 2: 200 µL MeOH (remove neutral and basic interferences) Elute: 50 µL ACN:IPA (40:60) with 5% FA Dilute: 125 µL H2O with 2% NH4OH (total volume of 175 µL results in a concentration factor of 2)
Ziling Lu, Jeffrey Mazzeo, Claude Mallet, Diane Diehl
Chemistry Applied Technology Group Waters Corporation, Milford, MA USA
OH
OH
COOH
OHOH
COOH
OH
O
ISTD: 8-Iso Prostaglandin E2
FW: 352.5 MRM: 351.0 → 315.0
Analyte: Prostaglandin F2a
FW: 354.5 MRM 353.0 → 193.0
HPLC CONDITIONS- Column: XTerra® MS C18, 2.1 x 30 mm, 3.5 µm Part Number: 186000398 Mobile Phase A: Water with 0.5% NH4OH Mobile Phase B: ACN with 0.5% NH4OH Flow Rate: 0.2 mL/min Gradient: Time Profile (min) %A %B 0.0 95% 5% 1.0 5% 95% Injection volume: 20 µL Temperature: Ambient Instrument: Alliance® 2795 Separsations Module RESULTS-
MS/MS CONDITIONS- Instrument: Waters Micromass® Quattro Ultima Source: Electrospray negative Source temperature: 150 ºC Desolvation temperature: 400 ºC Desolvation gas flow: 520 L/hr Collision gas cell: 2.0e-3 mbar Cone voltage: 60 V for both Collision energy: 25 eV for F2a and 12 eV for E2
0.5% NH4OH
0.5% NH4OH
0.5% FA
0.5% FA
F2a
F2a
ISTD
ISTD
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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%
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%
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2.09e43.31
2.440.450.120.24
1.220.60 0.74 2.391.962.05
3.12
3.34
3.59 3.73 4.974.13 4.47
351 > 315
4.12e53.37
0
100
%
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%
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1.50e52.99
351 > 315
1.52e62.99
0.5% NH4OH
0.5% NH4OH
0.5% FA
0.5% FA
F2a
F2a
ISTD
ISTD
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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%
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2.09e43.31
2.440.450.120.24
1.220.60 0.74 2.391.962.05
3.12
3.34
3.59 3.73 4.974.13 4.47
351 > 315
4.12e53.37
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2.09e4353 > 193
2.09e43.31
2.440.450.120.24
1.220.60 0.74 2.391.962.05
3.12
3.34
3.59 3.73 4.974.13 4.47
351 > 315
4.12e5
3.31
2.440.450.120.24
1.220.60 0.74 2.391.962.05
3.12
3.34
3.59 3.73 4.974.13 4.47
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1.50e5
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1.52e6
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1.52e62.99
Figure 1. Before developing the SPE method, we needed to develop an LC/MS/MS method for these analytes. We made an infusion of the samples into the MS to determine the MRM transitions and the optimum settings for the MS. We then ran the samples under low and high pH conditions using an XTerra® MS C18 column. Under high pH conditions with NH4OH, we found the best signal-to-noise and sensitivity for the analytes and chose that as our final method.
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QC_NH3_Weighted_5 MRM of 2 Channels ES-353 > 193
3.56e52.92
12482
3.2123
3.4779
3.6625
QC_NH3_Weighted_5 MRM of 2 Channels ES-351 > 315
3.06e62.93
112520
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-353 > 193
3.98e52.95
14435
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-351 > 315
2.67e62.95
103163
2.5376
0.2628
3.1393
Standard Solution, not taken through SPE
Standard Solution, taken through SPE
F2a
F2a
ISTD
ISTD0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time0
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QC_NH3_Weighted_5 MRM of 2 Channels ES-353 > 193
3.56e52.92
12482
3.2123
3.4779
3.6625
QC_NH3_Weighted_5 MRM of 2 Channels ES-351 > 315
3.06e62.93
112520
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-353 > 193
3.98e52.95
14435
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-351 > 315
2.67e62.95
103163
2.5376
0.2628
3.1393
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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QC_NH3_Weighted_5 MRM of 2 Channels ES-353 > 193
3.56e5
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100
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QC_NH3_Weighted_5 MRM of 2 Channels ES-353 > 193
3.56e52.92
12482
3.2123
3.4779
3.6625
QC_NH3_Weighted_5 MRM of 2 Channels ES-351 > 315
3.06e6
2.9212482
3.2123
3.4779
3.6625
QC_NH3_Weighted_5 MRM of 2 Channels ES-351 > 315
3.06e62.93
112520
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-353 > 193
3.98e5
2.93112520
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-353 > 193
3.98e52.95
14435
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-351 > 315
2.67e6
2.9514435
MAX_STDinH2O_NH4OH_98 MRM of 2 Channels ES-351 > 315
2.67e62.95
103163
2.5376
0.2628
3.1393
Standard Solution, not taken through SPE
Standard Solution, taken through SPE
F2a
F2a
ISTD
ISTD
Figure 2. Since these analytes are acids, we chose to use the Oasis® MAX (mixed-mode anion exchange) SPE sorbent for our clean-up step. This sorbent l is highly selective for the analytes and allowed us to obtain an extremely clean extract. Additionally, in order to pre-concentrate the sample and to eliminate the evaporation and reconstitution steps, we utilized the Oasis® µElution Plate which enables low elution volumes. We first tried the generic SPE method that is recommended for the MAX material on a sample dissolved in water. We obtained recoveries of 115% and 92% for the F2a and ISTD, respectively.
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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MAX_STDinH2O_NH4OH_6 MRM of 2 Channels ES-353 > 193
1.27e5Area
2.964200
MAX_STDinH2O_NH4OH_5 MRM of 2 Channels ES-353 > 193
6.71e4Area
2.962129
0.6034
3.0643
MAX_STDinH2O_NH4OH_4 MRM of 2 Channels ES-353 > 193
3.84e4Area
2.971139
2.3536
0.2328
MAX_STDinH2O_NH4OH_3 MRM of 2 Channels ES-353 > 193
1.81e4Area
2.96458
MAX_STDinH2O_NH4OH_2 MRM of 2 Channels ES-353 > 193
1.27e4Area
2.962180.1 pg/µL
0.2 pg/µL
0.5 pg/µL
1 pg/µL
2 pg/µL
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MAX_STDinH2O_NH4OH_6 MRM of 2 Channels ES-353 > 193
1.27e5Area
2.964200
MAX_STDinH2O_NH4OH_5 MRM of 2 Channels ES-353 > 193
6.71e4Area
2.962129
0.6034
3.0643
MAX_STDinH2O_NH4OH_4 MRM of 2 Channels ES-353 > 193
3.84e4Area
2.971139
2.3536
0.2328
MAX_STDinH2O_NH4OH_3 MRM of 2 Channels ES-353 > 193
1.81e4Area
2.96458
MAX_STDinH2O_NH4OH_2 MRM of 2 Channels ES-353 > 193
1.27e4Area
2.96218
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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%
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MAX_STDinH2O_NH4OH_6 MRM of 2 Channels ES-353 > 193
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%
MAX_STDinH2O_NH4OH_6 MRM of 2 Channels ES-353 > 193
1.27e5Area
1.27e5Area
2.964200
MAX_STDinH2O_NH4OH_5 MRM of 2 Channels ES-353 > 193
6.71e4Area
2.964200
MAX_STDinH2O_NH4OH_5 MRM of 2 Channels ES-353 > 193
6.71e4Area
2.962129
0.6034
3.0643
MAX_STDinH2O_NH4OH_4 MRM of 2 Channels ES-353 > 193
3.84e4Area
2.962129
0.6034
3.0643
MAX_STDinH2O_NH4OH_4 MRM of 2 Channels ES-353 > 193
3.84e4Area
2.971139
2.3536
0.2328
MAX_STDinH2O_NH4OH_3 MRM of 2 Channels ES-353 > 193
1.81e4Area
2.971139
2.3536
0.2328
MAX_STDinH2O_NH4OH_3 MRM of 2 Channels ES-353 > 193
1.81e4Area
2.96458
MAX_STDinH2O_NH4OH_2 MRM of 2 Channels ES-353 > 193
1.27e4Area
2.96458
MAX_STDinH2O_NH4OH_2 MRM of 2 Channels ES-353 > 193
1.27e4Area
2.962180.1 pg/µL
0.2 pg/µL
0.5 pg/µL
1 pg/µL
2 pg/µL
0.1 pg/µL
0.2 pg/µL
0.5 pg/µL
1 pg/µL
2 pg/µL
Figure 3. Calibration curve date for the Prostaglandin standards, our calibration range is 0.1 to 100 pg/µL. Our LOD is 0.1 pg/µL.
5 pg/µL
10 pg/µL
20 pg/µL
50 pg/µL
100 pg/µL
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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%
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MAX_STDinH2O_NH4OH_11 MRM of 2 Channels ES-353 > 193
5.52e6Area
2.95195484
MAX_STDinH2O_NH4OH_10 MRM of 2 Channels ES-353 > 193
2.69e6Area
2.9694444
3.1569
MAX_STDinH2O_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.17e6Area
2.9540790
0.4729
0.2344
MAX_STDinH2O_NH4OH_8 MRM of 2 Channels ES-353 > 193
6.16e5Area
2.9622223
MAX_STDinH2O_NH4OH_7 MRM of 2 Channels ES-353 > 193
3.19e5Area
2.9610840
5 pg/µL
10 pg/µL
20 pg/µL
50 pg/µL
100 pg/µL
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MAX_STDinH2O_NH4OH_11 MRM of 2 Channels ES-353 > 193
5.52e6Area
2.95195484
MAX_STDinH2O_NH4OH_10 MRM of 2 Channels ES-353 > 193
2.69e6Area
2.9694444
3.1569
MAX_STDinH2O_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.17e6Area
2.9540790
0.4729
0.2344
MAX_STDinH2O_NH4OH_8 MRM of 2 Channels ES-353 > 193
6.16e5Area
2.9622223
MAX_STDinH2O_NH4OH_7 MRM of 2 Channels ES-353 > 193
3.19e5Area
2.9610840
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MAX_STDinH2O_NH4OH_11 MRM of 2 Channels ES-353 > 193
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MAX_STDinH2O_NH4OH_11 MRM of 2 Channels ES-353 > 193
5.52e6Area
5.52e6Area
2.95195484
MAX_STDinH2O_NH4OH_10 MRM of 2 Channels ES-353 > 193
2.69e6Area
2.95195484
MAX_STDinH2O_NH4OH_10 MRM of 2 Channels ES-353 > 193
2.69e6Area
2.9694444
3.1569
MAX_STDinH2O_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.17e6Area
2.9694444
3.1569
MAX_STDinH2O_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.17e6Area
2.9540790
0.4729
0.2344
MAX_STDinH2O_NH4OH_8 MRM of 2 Channels ES-353 > 193
6.16e5Area
2.9540790
0.4729
0.2344
MAX_STDinH2O_NH4OH_8 MRM of 2 Channels ES-353 > 193
6.16e5Area
2.9622223
MAX_STDinH2O_NH4OH_7 MRM of 2 Channels ES-353 > 193
3.19e5Area
2.9622223
MAX_STDinH2O_NH4OH_7 MRM of 2 Channels ES-353 > 193
3.19e5Area
2.9610840
Figure 4. Calibration curve date for the Prostaglandin standards, our calibration range is 0.1 to 100 pg/µL. Our LOD is 0.1 pg/µL.
Coefficient of Determination: 0.997131Calibration curve: 2004.29 * x + 71.7943Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0ng/mL0
2.06e5
Resp
onse
Coefficient of Determination: 0.997131Calibration curve: 2004.29 * x + 71.7943Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Coefficient of Determination: 0.997131Calibration curve: 2004.29 * x + 71.7943Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0ng/mL0
2.06e5
Resp
onse
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2.06e5
Resp
onse
Figure 5. We then generated a calibration curve for the standards to determine our concentration range and our detection limits. Our calibration range is 0.1 to 100 pg/µL. Our LOD is 0.1 pg/µL.
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MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-353 > 193
8.13e32.052.001.851.010.730.12 0.600.16 0.58 1.04 1.501.29
3.062.13 3.012.322.842.57 3.11 3.27 3.47
3.953.89 4.804.034.454.12 4.48
MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-351 > 315
1.90e62.98
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-353 > 193
6.47e52.95
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-351 > 315
6.38e62.95
3.09
F2a, H2O
F2a, Plasma
ISTD, H2O
ISTD, Plasma
The rat plasma contained about 5 pq/uL F2a naturally.
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100
%
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MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-353 > 193
8.13e32.052.001.851.010.730.12 0.600.16 0.58 1.04 1.501.29
3.062.13 3.012.322.842.57 3.11 3.27 3.47
3.953.89 4.804.034.454.12 4.48
MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-351 > 315
1.90e62.98
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-353 > 193
6.47e52.95
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-351 > 315
6.38e62.95
3.09
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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%
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
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100
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100
%
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100
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MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-353 > 193
8.13e3
0
100
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MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-353 > 193
8.13e32.052.001.851.010.730.12 0.600.16 0.58 1.04 1.501.29
3.062.13 3.012.322.842.57 3.11 3.27 3.47
3.953.89 4.804.034.454.12 4.48
MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-351 > 315
1.90e6
2.052.001.851.010.730.12 0.600.16 0.58 1.04 1.501.293.062.13 3.012.32
2.842.57 3.11 3.27 3.473.953.89 4.804.03
4.454.12 4.48
MAX_STDinH2O_NH4OH_1 MRM of 2 Channels ES-351 > 315
1.90e62.98
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-353 > 193
6.47e5
2.98
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-353 > 193
6.47e52.95
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-351 > 315
6.38e6
2.95
MAX_RatPlasma_NH4OH_1 MRM of 2 Channels ES-351 > 315
6.38e62.95
3.09
F2a, H2O
F2a, Plasma
ISTD, H2O
ISTD, Plasma
The rat plasma contained about 5 pq/uL F2a naturally.
20 pg/ul
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
100
%
0
100
%
0
100
%
0
100
%
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-353 > 193
1.46e62.95
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-351 > 315
2.54e62.95
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.59e62.94
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-351 > 315
6.43e62.95
3.08
F2a
ISTD
F2a
ISTD
Sample in water
Sample in water
Sample in plasma
Sample in plasma
20 pg/ul
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
100
%
0
100
%
0
100
%
0
100
%
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-353 > 193
1.46e62.95
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-351 > 315
2.54e62.95
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.59e62.94
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-351 > 315
6.43e62.95
3.08
20 pg/ul
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
100
%
20 pg/ul
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-353 > 193
1.46e6
0
100
%
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-353 > 193
1.46e62.95
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-351 > 315
2.54e6
2.95
MAX_STDinH2O_NH4OH_100 MRM of 2 Channels ES-351 > 315
2.54e62.95
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-
2.95
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-353 > 193
1.59e6353 > 193
1.59e62.94
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-351 > 315
6.43e6
2.94
MAX_RatPlasma_NH4OH_9 MRM of 2 Channels ES-351 > 315
6.43e62.95
3.08
F2a
ISTD
F2a
ISTD
Sample in water
Sample in water
Sample in plasma
Sample in plasma
Figure 6. We then ran blank plasma through the SPE procedure and found that the blank plasma contains about 5 pg/µL of F2a.
Figure 7. We spiked rat plasma with 20 pg/µL of F2a and ran both the standard and the plasma through the clean up procedure and obtained good recoveries, after accounting for the naturally occurring F2a.
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CONCLUSIONS-
Using the generic SPE protocol on the Oasis® MAX µElution plate, we developed a high-throughput method for prostaglandin F2a with: -Good sample recoveries -Concentration factor at least 2x -A calibration range of 0.1 – 100 pg/µL -LOD of 0.1 pg/µL
