Plasma-free Metanephrines Quantitation with Automated Online Sample Preparation and a Liquid Chromatography-Tandem Mass Spectrometry Method

Transcription

1 Plasma-free Metanephrines Quantitation with Automated Online Sample Preparation and a Liquid Chromatography-Tandem Mass Spectrometry Method Xiang He and Marta Kozak ThermoFisher Scientific, San Jose, CA, USA

2 Overview Purpose: To develop and validate a fast and analytically sensitive LC-MS/MS method to quantify plasma-free metanephrines (Pmets) with Thermo Scientific TurboFlow technology for clinical research use. Methods: Plasma samples were processed with online sample preparation assisted with ion-pairing reagents and analyzed with porous graphitic carbonbased LC chromatography. Results: This method was linear from pg/ml for metanephrine and pg/ml for normetanephrine with an accuracy of 8.6% to 93.5% and 8.9% to 11.7%, respectively. Introduction Unconjugated metanephrine (MN) and normetanephrine (NMN) in plasma, collectively known as Pmets (Figure 1), are widely used biomarkers for pheochromocytoma for clinical research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become widely used to measure Pmets because of its high analytical specificity and less laborious sample preparation. Thermo Scientific TurboFlow technology is an automated online sample preparation technique that has been coupled to LC-MS/MS for the quantitative analysis of biological samples. Recently, we have reported an LC-MS/MS method for measuring Pmets using ion-pairing SPE (IP-SPE) and porous graphitic carbon (PGC) column chromatography. Our goal is to develop a method to measure plasma-free metanephrines and reduce method time while maintaining analytical performance compared to the original offline SPE method. Figure 1. Molecular structures of metanephrine and normetanephrine. O OH H N O OH NH 2 HO Methods Metanephrine Normetanephrine A.5-mL sample (charcoal stripped serum: CSS; K 2 EDTA plasma) was spiked with internal standards (IS) and then mixed with.25 ml of 1% trichloroacetic acid (w/v) in water (Figure 2). The mixture was vortexed and stored at C for 3 min. The mixture was then centrifuged at 16, g for 1 min, and 1 µl of supernatant was injected for LC-MS/MS analysis. The TurboFlow method with automated online sample preparation was performed with a Thermo Scientific TurboFlow Cyclone MCX-2 column. Perfluoroheptanoic acid (PFHA) was used as the ion-pair during the online sample preparation. Analytical separation was carried out on a Thermo Scientific Hypercarb column (5 3 mm, 5.-μm particle size) at 7 C. The total LC runtime was 12 min (Table 1). A Thermo Scientific TSQ Vantage triple stage quadrupole mass spectrometer was operated with a heated electrospray ionization (HESI-II) source in positive ionization mode. Data were acquired in the selected reaction monitoring (SRM) mode (Table 2). Figure 2. Sample preparation workflow. 1. Add sample, spike, IS. HO 4. 3 min at C. 5. Centrifuge. TurboFlow LC-MS/MS 2. Vortex 3. Add 1% TCA. (1/2 v) 2 Plasma-free Metanephrines Quantitation with Automated Online Sample Preparation and a Liquid Chromatography-Tandem Mass Spectrometry Method

3 Figure 3. Focus Technical Mode diagram of TurboFlow technology. Load Transfer MCX-2 Waste MCX-2 Waste LP EP LP EP Transfer Loop MS Hypercarb Transfer Loop MS Hypercarb Wash/Elute MCX-2 Waste LP EP Transfer Loop MS Hypercarb Table 1. LC gradient (Column: Hypercarb mm, 5 μm, 7 C). Loading Eluting Loading: A:.1% PFHA in water; B: 6% ACN in water; C: mixture of isopropanol, CAN, and acetone (1:1:1 v/v/v) with.3% formic acid; D: 5 mm NH 4 Ac and 5% ACN in water; Eluting: A: 5 mm NH 4 FA and 1% formic acid in water; B:.1% formic acid in ACN; C: mixture of isopropanol, AACN, and acetone (9:9:2 v/v/v); D:.1% PFHA in water. Eluting LC column temperature: 7 C Table 2. Source parameters and SRM transitions. Ionization HESI-II Vaporizer temp ( C) 48 Capillary temp ( C) 38 Spray voltage (V) 15 Sheath gas (AU) 3 Auxillary gas (AU) 5 Data acquisition mode SRM Chrom filter peak width (s) 5 Collision gas pressure (mtorr) 1 Scan width (m/z).1 Scan time (s).1 Q1 (FWMH).7 Q3 (FWMH).7 Compound Q1 Q3 CE S-Lens MN NMN MN-d NMN-d Validation Calibration curves were generated with CSS spiked with MN and NMN. MN-d 3 and NMN-d 3 were used as internal standards. Individual human K 2 EDTA plasma was evaluated and compared to CSS. Assay accuracy and precision were evaluated by using two levels of quality control samples prepared in CSS. Extraction recovery was assessed by comparing peak area of TurboFlow injection to direct analytical column injection of MN, NMN, MN-d 3 and NMN-d 3 spiked in mobile phase (n=3). Ion suppression was evaluated by: 1) post-column T infusion of deuterated MN and NMN and 2) comparing the MS responses of MN-d 3 and NMN-d 3 in mobile phase buffer (blank, n=4) and individual human plasma samples (n=4) at the same concentrations (4 pg/ml for both MN-d 3 and NMN-d 3 ). Epinephrine shares the same SRM transitions as normetanephrine and was checked for interference. Assay linearity was determined by serial dilution of spiked CSS with blank CSS. Triplicates were run for each concentration level. Thermo Scientific Poster Note PN63585_E 6/12S 3

4 Results Calibration Curve Calibration curves of both MN and NMN (Figure 4) in CSS showed excellent linearity. Results also indicated no endogenous levels of MN and NMN in blank CSS. Figure 4. Calibration curves MN (A) and NMN (B) in CSS. 1.9 MN Y = X R 2 =.9992 W: 1/X Area Ratio Concentration 1.2 NMN Y = X R 2 =.9988 W: 1/X Area Ratio Concentration Recovery Absolute recovery of MN, NMN, and their IS ranged from 56.4% to 62.4%, and the relative recovery of MN and NMN was 9.9% and 97.8%, respectively (Table 3). Table 3. Recovery of MN, NMN and their IS. Online Extraction (mean ± CV) b Direct Injection (mean ± CV) Absolute Recovery Relative Recovery MN (5 pg/ml) a 6281 ± 2.7% ± 1.5% NMN (25 pg/ml) a ± 5.6% ± 9.4% MN-d3 (5 pg/ml) a 4716 ± 1.1% 6679 ± 11.4% 61. NMN-d3 (5 pg/ml) a ± 3.7% ± 11.8% 62.4 a MN, NMN, MN-d 3 and NMN-d 3 were spiked to MPA at specified concentration levels. b Measured peak area with CV (n=2) 4 Plasma-free Metanephrines Quantitation with Automated Online Sample Preparation and a Liquid Chromatography-Tandem Mass Spectrometry Method

5 Ion Suppression We used two tests to evaluate ionization suppression. In the first test (post-column infusion), no obvious ionization suppression was detected in the SRM chromatograms of MN-d 3 and NMN-d 3 using processed human plasma samples compared to injections of mobile phase blanks. In the second test, the average MS responses (integrated area) of MN-d 3 and NMN-d 3 from mobile phase blank and real human plasma samples were calculated and the intensity ratio with standard deviation between human plasma (n=4) and mobile phase blank (n=4) was 113.3% ± 18.4% and 126.4%% ± 18.% for MN-d 3 and NMN-d 3, respectively. This result was in line with the first test, but apparently, the second test rendered a more accurate quantitative evaluation of the ionization suppression or enhancement. We concluded that this method has no obvious ionization suppression or enhancement. Linearity Results from the 24 linearity samples (8 levels, n=3) are summarized in Table 4. After LLOQ and HLOQ were established, the linearity was determined to be pg/ml for MN and pg/ml for NMN. Within the linear range, accuracy ranged from 8.6% to 93.5% for MN, and from 8.9% to 11.7% for NMN (Table 4). CV (n=3) from all linearity levels ranged from 3.1% to 13.7% for MN, and from 1.6% to 1.7% for NMN. The determined LLOQ was 6.3 pg/ml for MN and 12.6 pg/ml for NMN. Table 4. Linearity. MN NMN Dilution factor Expected Measured CV of triplicates Accuracy Expected Measured CV of triplicates Accuracy Mean Stdev Precision Precision was first assessed with spiked charcoal stripped serum. Inter- and intraassay CV values at two levels of both analytes varied between 2.1% to 1.9% (Table 5). Precision was also assessed with a human plasma pool (35.6 pg/ml of MN and 53.1 pg/ml of NMN, n=5) and the determined intra-assay CV was 6.3% and 7.8% for MN and NMN, respectively (data not shown). Table 5. Accuracy and precision. MN NMN CSS 31 pg/ml 25 pg/ml 63 pg/ml 5 pg/ml Intra-assay Precision n= Accuracy Inter-assay Precision n= Accuracy Interference Epinephrine and normetanephrine share the same SRM transitions and could not be differentiated just by MS/MS analysis. Using the Hypercarb analytical column, the epinephrine peak was baseline resolved (.3 min apart) from the normetanephrine peak. Figure 5 shows a processed human plasma sample. Data Examples of Clinical Research Samples See Figure 5. Thermo Scientific Poster Note PN63585_E 6/12S 5

6 Figure 5. Representative SRM chromatograms of MN and NMN in a spiked CSS sample (top) and and individual human plasma sample (bottom). 398 Intensity NMN: m/z pg/ml MN: m/z pg/ml Time (min) 688 Intensity NMN: m/z pg/ml MN: m/z pg/ml Conclusion Time (min) A fast, automated, and analytically sensitive LC-MS/MS method was developed to quantify plasma metanephrines for clinical research purposes. The sample preparation procedure was significantly simplified compared to a previously reported offline IP-SPE method 1 by using online TurboFlow technology 2. The presence of PFHA during the online sample preparation was critical to the success of this method. 1. The total online extraction and analytical LC runtime was 12 minutes. 2. This method was linear from 6.3 to pg/ml for metanephrine and 12.6 to pg/ml for normetanephrine, with an accuracy of 8.6% to 93.5% and 8.9% to 11.7%, respectively. 3. The lower limit of quantitation was 6.3 pg/ml for metanephrine and 12.6 pg/ml for normetanephrine. 4. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.% to 1.5%. Overall, the analytical performance achieved with this automated online TurboFlow method is consistent with the previously reported offline SPE method 1. More importantly, the TurboFlow method significantly saved sample preparation time by more than 5% and eliminated the expense of SPE cartridges with an offline approach. References 1. He, X.; Gabler, J.; Yuan, C.; Wang, S.; Shi, Y.; and Kozak, M. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 211, 879(23), He, X.; and Kozak, M. Anal. Bioanal. Chem., 212, 42(9), For Research Use Only. Not for use in diagnostic procedures. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Plasma-free Metanephrines Quantitation with Automated Online Sample Preparation and a Liquid Chromatography-Tandem Mass Spectrometry Method

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