Determination of Plasma Renin Activity by LC/MS/MS for Clinical Research

Linda Côté, Agilent Technologies, Inc., Montréal, QC MSACL 2015 Poster #40

Introduction Experimental Results and Discussion

Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is ideally LC Analytical Method Accuracy and Reproducibility suited for the rapid analysis of analytes in complex matrices. A highly sensitive and Agilent 1290 HPLC binary pumps, well plate sampler with thermostat, temperature- specific LC/MS/MS analytical method has been developed for the determination of controlled column compartment. Bio-Rad Lyphocheck hypertension markers controls were used to test the accuracy and plasma renin activity (PRA) for clinical research. An analytical method for quantifying reproducibility of this analytical method. Measurements were repeated in triplicates to Angiotensin I in plasma is applied for the determination of Plasma Renin Activity. Parameter Value assess intra-day and on three separate day to assess inter-day reproducibility and CVs Plasma samples are incubated for 3 hours at 37oC. A sample preparation procedure by were found to be below 6%, see table 4. All measurements are in ng/L/s. Analytical Column Agilent Poroshell 120 SB-C18, 2.1 x 50 mm, 2.7 µm, PN: 689775-902 solid phase extraction (SPE) allows efficient extraction of Angiotensin I in plasma. Plasma renin activity is calculated by subtracting Angiotensin I concentration in a blank Guard Column Agilent Poroshell 120 SB-C18, 2.1 x 5 mm, 2.7 µm, PN: 821725-912 plate. Measured Measured Injection Volume 20 µl Intra-day Inter-day QC Value Value Needle Wash 1:1:1:1 MeOH:ACN:IPA:H2O + 0.1% formic acid in Flush port for 20 CV% CV% Renin Level Intra-day Inter-day seconds (n=3) (n=3) Angiotensinogen Angiotensin I Injector Temperature 4 °C (n=3) (n=3) pH = 6.0 Mobile Phase A Water + 0.2 % Formic Acid Plasma Renin 1 0.404 5.0 0.443 5.4 Figure 1. Renin-Angiotensin system Mobile Phase B Methanol + 0.2 % Formic Acid Activity 2 0.933 2.7 0.956 4.4 Flow rate 0.6 mL/min. ng/L/s 3 3.223 5.8 3.573 4.4 Plasma Renin Activity is then calculated by: Pump Gradient Time (min.) %B 0.0 10 Table 4. Results of Bio-Rad Lyphocheck hypertension markers controls by LC-MS/MS 0.5 10 PRA = ([Angiotensin I]37oC – [Angiotensin I]0oC) Δt 1.5 95 3.5 95 Calibration curve Calibrators were created by spiking a 1% bovine serum albumin buffer pH 6 solution Preparation of Angiotensin I calibrants was done in 1% bovine serum albumin and were with various concentrations of Angiotensin I. The chromatographic system consists of Stop time 3.5 min. prepared as zero samples. The procedure was repeated on three separate day to an Agilent Poroshell 120 SB-C18 column and a mobile phase comprised of methanol assess inter-day reproducibility and CVs were found to be below 6%. Accuracies at all and water containing 0.2% formic acid. Quantifier and qualifier transitions were Post Time 1.5 min. levels are within acceptance criteria, see table 5. monitored and an isotopically labelled Internal Standard was included to ensure Table 1. LC Parameters accurate and reproducible quantitation. Angiotensin I - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 0 QCs

x10 1 y = 0.091912 * x - 0.003043 R^2 = 0.99964284 Type:Linear, Origin:Ignore, Weight:1/x MS Analytical Method 0.95 0.9 Experimental 0.85 Agilent 6460 QQQ LC/MS with Agilent JetStream Technology 0.8

0.75 Ion Mode Agilent JetStream 0.7 0.65

Sample Preparation Electrospray, positive ionization 0.6 0.55 Drying gas 350°C, 7 L/min 0.5 Calibrators (Proteochem) are prepared with 1% bovine serum albumin buffer pH 6 0.45 solution. Isotopically labelled Internal standard (Anaspec) and Biorad plasma controls Nebulizer gas 40 psi 0.4 0.35 were used. Plasma samples should not exceed 4°C at any time until ready for Sheath gas 400°C, 12 L/min 0.3 incubation or extraction. 0.25 Capillary voltage 4000 V 0.2 0.15 0.1

Step 1: Preparation of generation and zero samples Nozzle voltage 0 V 0.05

0 Q1/Q3 resolution 0.7 unit -0.05 Generation samples Zero samples -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Concentration (ng/ml)

∆ EMV 200 V ative Responses 250 μL of plasma samples and controls 250 μL of plasma samples, calibrators Figure 3. Calibration curve for Angiotensin I, 0.17 to 100 ng/mL + 50 μL generation buffer* and controls Scan rate 3 spectra/sec Mix Table 2. MS Parameters

Compound Prec Ion Prod Ion Frag (V) CE (V) CAV (V) Incubate at 37°C for 3 hours Inter-day Angiotensin I (Quant) 152.1 107 115 16 5 R2 Concentration Accuracy (%) Compound CV (%) Angiotensin I (Qualfier) 176.1 111.1 65 24 5 n = 3 (ng/mL) n = 3 Angiotensin I - ISTD 190.1 172.1 70 8 5 n = 3 Add 300 μL of ISTD solution Add 300 μL of ISTD solution Table 3: MRM Transitions table 0.1688 114.3 3.6 (10 ng/mL in 10% formic acid) (10 ng/mL in 10% formic acid) 0.3375 101.0 3.8 Mix Mix 0.675 96.9 5.3 Results and Discussion 1.35 98.6 3.2 Angiotensin I 0.9996 Perform SPE procedure Perform SPE procedure 2.7 93.2 5.7 Angiotensin I Calibrator - 2.7 ng/mL Bio-Rad Control Level 1 - PRA 0.44 ng/L/s 9 99.2 0.8 + MRM CF=0.000 DF=0.000 (435.2394 -> 654.3683) 010_Cal6.d + MRM CF=0.000 DF=0.000 (435.2394 -> 654.3683) 019_QC1_incubation.d *: Generation buffer: 1M Tris Base+ 0.2M EDTA + 1 mM PMSF, pH 5.5 with acetic acid x10 3 x10 3 30 98.4 1.7

1.548 1.544 1.5 100 100.8 0.7 Step 2 - Solid Phase Extraction (SPE) 1.5 Angiotensin I - ISTD 1 Angiotensin I - ISTD 1 Table 5: Summary of analyte performance. 0.5 Use preparation from step 1 0.5

0 0 + MRM CF=0.000 DF=0.000 (432.9003 -> 647.3511) 010_Cal6.d + MRM CF=0.000 DF=0.000 (432.9003 -> 647.3511) 019_QC1_incubation.d 1: Condition SPE cartridge (Agilent BondElut Plexa, 30 mg, 3 mL, x10 2 x10 2

PN: 12109303) with: 1.545 8 1.545

1 mL methanol 3 Angiotensin I 6 Angiotensin I Conclusions 1 mL 5% formic acid in water Quantifier MRM 2 Quantifier MRM 4 2: Add samples 1 2 A robust analytical method for quantifying Angiotensin I in plasma by LC/MS/MS 0 + MRM CF=0.000 DF=0.000 (432.9003 -> 619.3000) 010_Cal6.d + MRM CF=0.000 DF=0.000 (432.9003 -> 619.3000) 019_QC1_incubation.d which is applied for the determination of Plasma Renin Activity has been developed. 3: Wash with: x10 2 x10 2 Typical analytical method performance results are well within acceptable criteria. 1 mL 5% formic acid in water 2 1.545 4 1.541 Angiotensin I 1 mL 20% methanol in water 1.5 3 Angiotensin I Dry at full vacuum for 5 minutes Qualifier MRM Qualifier MRM 2 References: 1 1 J Grace Van Der Gugten, Daniel T Holmes, St.Paul’s Hospital, University of British Columbia, Vancouver, Canada. “Plasma 4: Elute with: 0.5 Renin Activity by Tandem Mass Spectrometry Employing Analyte Immunoprotection”. ASMS 2012 Vancouver 250 µL of methanol. Poster. 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 Bystrom, Cory E. "Plasma Renin Activity by LC-MS/MS: Development of a Prototypical Clinical Assay Reveals a Apply vacuum 5” Hg for 60 seconds Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Subpopulation of Human Plasma Samples with Substantial Peptidase Activity“. Clinical Chemistry 56:10 1561–1569 (2010). DOI: 10.1373/clinchem.2010.146449 Note: use of plastic ware is recommended for optimum recoveries Figure 2. Chromatography for Angiotensin I Calibrator – 2.7 bg/mL and Bio-Rad Lyphocheck Control Level 1

For Research Use Only. Not for use in diagnostic procedures.